NOVEL RNAi THERAPEUTIC FOR TREATMENT OF HEPATITIS C INFECTION

ABSTRACT

Small interfering RNAs (siRNAs) or small hairpin RNA (shRNAs) and compositions comprising same are provided that target human cyclophilin A (CyPA) to inhibit Hepatitis C (HCV) infection. Such siRNA and shRNAs may have a length of from about 19 to about 29 contiguous nucleotides corresponding to a specific region of human cyclophilin A (CyPA) cDNA of from about nucleotide 155 to about nucleotide 183 having particular potency against CyPA and HCV. Such siRNA and shRNAs may be formulated as naked compositions or pharmaceutical compositions. DNA polynucleotides, plasmids, and viral or non-viral vectors are also provided that encode siRNA or shRNA molecules, which may be delivered directly to cells or in combination with delivery agents, such as lipids, polymers, encapsulated lipid particles, such as liposomes. Methods for treating, managing inhibiting, preventing, etc., HCV infection using such siRNA and shRNAs and compositions comprising same are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 12/167,402,filed Jul. 3, 2008, allowed, which claims the priority date from U.S.Prov. App. No. 60/948,040, entitled “NOVEL RNAi THERAPEUTIC FORTREATMENT OF HEPATITIS C INFECTION,” filed Jul. 5, 2007, and the entiredisclosure and contents of this provisional application are herebyincorporated by reference.

GOVERNMENT INTEREST STATEMENT

This invention was made with support from the State of Florida underFlorida Department of Health Grant No. 06NIR. The State of Florida mayhave rights to this invention.

BACKGROUND

1. Field of the Invention

The present invention relates generally to compositions comprising smallinterfering RNA (siRNA) or small hairpin RNA (shRNA) sequencescorresponding to at least a portion of cyclophilin A (CyPA) to treat,manage, inhibit, or prevent, etc., viral infection by hepatitis C virus(HCV) in a host. The present invention further generally relates tomethods of treating, managing, inhibiting, preventing, etc., HCV usingsuch compositions.

2. Background of the Invention

Hepatitis C virus (HCV) is a major human pathogen, infecting anestimated 170 million persons worldwide—roughly five times the numberinfected by human immunodeficiency virus type 1. A substantial fractionof these HCV infected individuals develop serious progressive liverdisease, including cirrhosis and hepatocellular carcinoma. HCV, a memberof the Flaviviridae family that includes other major human pathogenssuch as dengue and West Nile viruses, contains a positive-strand RNAgenome of 9.6 kb encoding a single polyprotein, which is processedthrough proteolysis to become at least 10 viral proteins. See, e.g.,Lindenbach, B. D. et al., “Unraveling hepatitis C virus replication fromgenome to function,” Nature 436:933-938 (2005), the entire disclosureand contents of which is hereby incorporated by reference.

RNA interference (“RNAi”) refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs). See, Fire et al., Nature, 391:806 (1998). Thecorresponding process in plants is commonly referred to aspost-transcriptional gene silencing or RNA silencing and may also bereferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes and iscommonly shared by diverse flora and phyla. See, Fire et al.,“RNA-triggered gene silencing,” Trends Genet. 15(9):358-63 (1999). Suchprotection from foreign gene expression may have evolved in response tothe production of double-stranded RNAs (dsRNAs) derived from viralinfection or from the random integration of transposon elements into ahost genome via a cellular response that specifically destroyshomologous single-stranded RNA or viral genomic RNA.

The process of RNAi begins by the presence of dsRNA in a cell, whereinthe dsRNA comprises a sense RNA having a sequence homologous to thetarget gene mRNA and an antisense RNA having a sequence complementary tothe sense RNA. In general, the presence of dsRNA stimulates the activityof a ribonuclease III enzyme referred to as Dicer. Dicer is involved inthe processing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs). See, e.g., Bernstein et al., “Role for abidentate ribonuclease in the initiation step of RNA interference,”Nature, 409: 363 (2001). Short interfering RNAs derived from Diceractivity are typically about 21 to about 23 nucleotides in length andcomprise about 19 base pair duplexes. See, e.g., Elbashir, S. M. et al.,“RNA interference is mediated by 21- and 22-nucleotide RNAs,” Genes Dev.15:188 (2001). The siRNAs in turn stimulate the RNA-induced silencingcomplex (RISC) by incorporating one strand of siRNA into the RISC anddirecting the degradation of the homologous mRNA target.

SUMMARY

According to a first broad aspect of the present invention, there isprovided a small interfering RNA (siRNA) comprising a sense RNA sequenceand an antisense RNA sequence, wherein the sense RNA sequence is atleast about 70% homologous to at least 19 contiguous nucleotides betweennucleotides 155 and 183 of human cyclophilin A sequence (SEQ ID NO: 1)and wherein the antisense RNA sequence is complementary to the sense RNAsequence.

According to a second broad aspect of the present invention, there isprovided a siRNA comprising a sense RNA sequence and an antisense RNAsequence, wherein the antisense RNA sequence is at least about 70%complementary to at least 19 contiguous nucleotides between nucleotides155 and 183 of human cyclophilin A sequence (SEQ ID NO: 1) and whereinthe sense RNA sequence is complementary to the antisense RNA sequence.

According to a third broad aspect of the present invention, there isprovided a DNA polynucleotide comprising a DNA sequence region encodinga sense RNA sequence that is at least about 70% homologous to at least19 contiguous nucleotides between nucleotides 155 and 183 of humancyclophilin A sequence (SEQ ID NO: 1).

According to a fourth broad aspect of the present invention, there isprovided a DNA polynucleotide comprising a DNA sequence region encodingan antisense RNA sequence that is at least about 70% complementary to atleast 19 contiguous nucleotides between nucleotides 155 and 183 of humancyclophilin A sequence (SEQ ID NO: 1).

According to a fifth broad aspect of the present invention, there isprovided a method for inhibiting Hepatitis C virus (HCV) infection,comprising the following steps: (i) administering at least one siRNAcomprising a sense RNA sequence and an antisense RNA sequence to anindividual infected or at risk of infection with HCV, and (ii)monitoring the level of HCV infection, wherein the sense RNA sequence isat least 70% homologous to at least 19 contiguous nucleotides betweennucleotides 155 and 183 of human cyclophilin A sequence (SEQ ID NO: 1)and wherein the antisense RNA sequence is complementary to the sense RNAsequence.

According to a sixth broad aspect of the present invention, there isprovided a method for inhibiting Hepatitis C virus (HCV) infectioncomprising the following steps: (i) administering at least one smallhairpin RNA (shRNA) comprising a sense RNA sequence and an antisense RNAsequence covalently linked by a hairpin sequence to an individualinfected or at risk of infection with HCV, and (ii) monitoring the levelof HCV infection, wherein the sense RNA sequence is at least about 70%homologous to at least 19 contiguous nucleotides between nucleotides 155and 183 of human cyclophilin A sequence (SEQ ID NO: 1) and wherein theantisense RNA sequence is complementary to the sense RNA sequence.

According to a seventh broad aspect of the present invention, there isprovided a method for inhibiting Hepatitis C virus (HCV) infectioncomprising the following steps: (i) administering at least one DNApolynucleotide comprising a DNA sequence region encoding a sense RNAsequence to an individual infected or at risk of infection with HCV, and(ii) monitoring the level of HCV infection, wherein the sense RNAsequence is at least about 70% homologous to at least 19 contiguousnucleotides between nucleotides 155 and 183 of human cyclophilin Asequence (SEQ ID NO: 1).

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described in conjunction with the accompanyingdrawings, in which:

FIG. 1A. is an image of Western blots showing expression of the proteinsNS5A and Actin in GS5 and RS2 replicon cells after being treated withCsA at various concentrations for 4 days;

FIG. 1B is an image of Western blots showing expression of the proteinsNS5A, CyPB, and CyPA in GS5 and RS2 replicon cells in response tovarious shRNAs (sh-Luc, sh-A159, sh-B710, or shi-C454) expressed forseven days prior to lysis;

FIG. 1C is a bar graph illustrating suppression of CyPC RNA expressionby sh-C454, relative to sh-Luc, as determined by real time RT-PCR, inGS5 and RS2 cells;

FIG. 1D is an image of plates containing GS5 and RS2 cells stained withviolet blue after introduction of various shRNAs (sh-Luc, sh-A159,sh-B710, or sh-C454) into the cells and following double selection withpuromycin and G418 and staining with violet blue.

FIG. 1E is an image of an RNA gel showing ³²P-labeled products of an invitro replication reaction of GS5 or RS2 lysate allowed to progress inthe presence of either a IgG negative control or an anti-CyPA antibodyfor 4 hours;

FIG. 2A is a schematic representation of the target sites of variousshRNAs (A159, A285, A285′, and A459) in CyPA, as well as target sites ofpreviously reported siRNAs, with example target sequence for each shownas antisense RNA sequences (A159 (SEQ ID NO. 4), A285 (SEQ ID NO. 20),A285′ (SEQ ID NO. 21), and A459 (SEQ ID NO. 22));

FIG. 2B is an image of Western blots showing expression of the proteinsNS5A, Actin, and CyPA in GS5 cells transduced with various shRNAs(sh-Luc, sh-A159, sh-A285, sh-A459, sh-NTC, or sh-A285′) for 7 daysprior to lysis;

FIG. 2C is an image of Western blots showing expression of the indicatedproteins (NS5A, Actin, and CyPA) in GS5 cells seven days aftertransfection with siRNA duplexes (si-NTC, si-A159, or si-285);

FIG. 2D is an image of Western blots showing expression of the proteinsNS5A, Actin, and Myc-tagged CyPA in GS5 cells transduced with sh-Luc orsh-A159, with or without transfection of a Myc-tagged CyPA cDNA (Myc-A#)containing silent mutations in the recognition site of sh-A159;

FIG. 2E is a bar graph showing relative HCV RNA expressed as a ratio ofHCV RNA extracted at day 4 to HCV RNA extracted at 7 hours afterco-electroporation of in vitro transcribed Rep1b RNA intoCyPA-knock-down Huh-7.5 cells, with or without expression from a CyPA#cDNA (Myc-A#) plasmid, with HCV RNA measured as a normalized level ofHCV IRES to GAPDH RNA. A parallel electroporation of Rep1b RNA intoHuh-7.5/sh-Luc cells serves as a positive control, which is set at 100%;

FIG. 3A is a graphical plot of the relative percentage of NS5A-GFPexpression normalized to untreated sample from RS2 cells and twoderivative cell lines stably expressing sh-Luc or sh-A159 and treatedwith increasing amounts of CsA for 4 days before being fixed for FACSanalysis;

FIG. 3B is an image of Western blots showing expression of the proteinsNS5A, Actin, and CyPA in RS2 cells expressing sh-Luc or sh-A159following treatment with varying concentrations of CsA;

FIG. 3C is a bar graph showing the relative percentage of NS5A-GFPexpressing RS2 cells transduced with either sh-Luc or sh-A159 for fourdays, with or without treatment with 0.5 μg/ml CsA for an additional 3days, as determined by FACS analysis;

FIG. 4A is an image of a Western blot showing expression of the proteinsKu80, CyPB, and CyPA in Huh-7.5 cells transduced with several shRNAs(sh-Luc, sh-A159, or sh-B710);

FIG. 4B is a bar graph showing normalized levels of CyPC RNA to GAPDHRNA in Huh-7.5 cells transduced with indicated shRNAs (sh-Luc orsh-C454);

FIG. 4C is an image of plates containing stained Huh-7.5 cellstransduced with several shRNAs (sh-Luc, sh-A159, sh-B710, or sh-C454)after four weeks following electroporation with one microgram of eitherH77 (GT 1a) or Con1 (GT 1b) replicons;

FIG. 4D is an image of Western blots showing expression of the proteinsNS5A, CyPB, and CyPA in H77 (GT 1a) and Con1 (GT 1b) replicon cellstransduced with the indicated shRNAs (sh-Luc or sh-A161);

FIG. 5A is an image of an RNA gel showing HCV IRES RNA levels relativeto cellular RNA helicase A (RHA) RNA in total RNA extracts from Huh7.5cells transduced with several shRNAs (sh-Luc, sh-A159, or sh-B710)following infection with HCVcc/JFH-1 for nine days;

FIG. 5B is a fluorescence microscopy image showing anticore antibody andDAPI (4′,6′-diamidino-2-phenylindole) staining of paraformaldehyde fixedHuh7.5 cells transduced with either sh-Luc or sh-A159 followinginfection with HCVcc/JFH-1 for nine days;

FIG. 5C is an image of Western blots showing expression of the proteinsNS3, GAPDH, and CyPA in total protein extracts from Huh7.5 cellstransduced with the indicated shRNAs (sh-Luc or sh-A159) at 0, 9, or 12days after infection with HCVcc/JFH-1 replicons;

FIG. 5D is an image of infection plaques formed by vesicular stomatitisvirus (VSV) in plates containing Huh7.5 cells, with or withouttransduction with sh-A159, and subjected to serial dilutions of 50 to5×10⁴ PFU of VSV;

FIG. 5E is an image of a Western blot showing expression of the proteinsNS3, CyPB, and CyPA in total protein extracts from Huh-7.5 cells tendays after transduction with the indicated shRNAs (sh-Luc or sh-A159)with the indicated transduction occurring after infection withHCVcc/JFH-1 for ten days;

FIG. 6A is an image of an RNA gel showing HCV IRES RNA from GS5 repliconcells co-precipitated with an antibody for CyPA or control IgG asdetected by RT-PCR;

FIG. 6B is an image of an RNA gel showing presence of HCV IRES RNA intotal RNA from GS5 and RS2 replicon cells and RNA from GS5 and RS2replicon cells co-precipitated with an antibody for CyPA in the presenceor absence of 4 μg/ml CsA;

FIG. 6C is an image of a Western blot showing presence of NS5B proteinin samples from GS5 replicon lysates incubated with equal amounts of GSTor GST-CyPA and purified with glutathione beads (FT=flowthrough;FW=final wash; and B=bound);

FIG. 6D is an image of a Western blot showing presence of NS5B insamples using the GST pull-down assay from FIG. 6C on lysates from GS5or RS2 cells in the presence of increasing amounts of CsA;

FIG. 6E is an image of a Western blot showing presence of NS5B and CyPAin total cell lysates from 293-T cells or samples produced byimmunoprecipitation with anti-Flag antibody of lysates from 293-T cellsfollowing co-transfection of 293-T cells with plasmids expressingFlag-tagged CyPA, with or without plasmids expressing Con1 NS5B for 48hours;

FIG. 7 is a bar graph showing the relative expression levels of CyPA,CyPB, and CyPC RNA in GS5 and RS2 replicon cells using real time PCR.

BRIEF DESCRIPTION OF SEQUENCE LISTINGS

SEQ ID NO: 1 (CyPA cDNA clone Y00052) shows the nucleotide sequence ofhuman cyclophilin A cDNA as provided by accession number Y00052 or asfollows:

GTGTACTATTAGCCATGGTCAACCCCACCGTGTTCTTCGACATTGCCGTCGACGGCGAGCCCTTGGGCCGCGTCTCCTTTGAGCTGTTTGCAGACAAGGTCCCAAAGACAGCAGAAAATTTTCGTGCTCTGAGCACTGGAGAGAAAGGATTTGGTTATAAGGGTTCCTGCTTTCACAGAATTATTCCAGGGTTTATGTGTCAGGGTGGTGACTTCACACGCCATAATGGCACTGGTGGCAAGTCCATCTATGGGGAGAAATTTGAAGATGAGAACTTCATCCTAAAGCATACGGGTCCTGGCATCTTGTCCATGGCAAATGCTGGACCCAACACAAATGGTTCCCAGTTTTTCATCTGCACTGCCAAGACTGAGTGGTTGGATGGCAAGCATGTGGTGTTTGGCAAAGTGAAAGAAGGCATGAATATTGTGGAGGCCATGGAGCGCTTTGGGTCCAGGAATGGCAAGACCAGCAAGAAGATCACCATTGCTGACTGTGGACAACTCGAATAAGTTTGACTTGTGTTTTATCTTAACCACCAGATCATTCCTTCTGTAGCTCAGGAGAGCACCCCTCCACCCCATTTGCTCGCAGTATCCTAGAATCTTTGTGCTCTCGCTGCAGTTCCCTTTGGGTTCCATGTTTTCCTTGTTCCCTCCCATGCCTAGCTGGATTGCAGAGTTAAGTTTATGATTATGAAATAAAAACTAAATAACAATTGTC;

SEQ ID NO: 2 (sense A-159) shows an example of a sense RNA sequence usedin embodiments of the present invention corresponding to nucleotides 159through 179 of human CyPA cDNA and is as follows: 5′-AAG GGU UCC UGC UUUCAC AGA-3′;

SEQ ID NO: 3 (sh-A159) shows an example of a shRNA sequence used inembodiments of the present invention corresponding to a sense sequencehaving nucleotides 159 through 179 of CyPA cDNA and is as follows:5′-AAG GGU UCC UGC UUU CAC AGA UUC AAG AGA UCU GUG AAA GCA GGA ACCCUU-3′;

SEQ ID NO: 4 (antisense A-159) shows an example of an antisense RNAsequence used in embodiments of the present invention complementary to asequence corresponding to nucleotides 159 through 179 of human CyPA cDNAand is as follows: 5′-UCU GUG AAA GCA GGA ACC CUU-3′;

SEQ ID NO: 5 (sh-A159 DNA template) shows an example of a minimal DNAtemplate for expression of a shRNA used in embodiments of the presentinvention and is as follows: 5′-TTC CCA AGG ACG AAA GTG TCT AAG TTC TCTAGA CAC TTT CGT CCT TGG GAA-3′;

SEQ ID NO: 6 (A-159) shows a sense DNA sequence for a siRNA or shRNA andis as follows: 5′-AAG GGT TCC TGC TTT CAC AGA-3′;

SEQ ID NO: 7 (A-285) shows a sense DNA sequence for a siRNA or shRNA andis as follows: 5′-AAG CAT ACG GGT CCT GGC ATC-3′;

SEQ ID NO: 8 (A-285′) shows a sense DNA sequence for a siRNA or shRNAand is as follows: 5′-AAG CAT ACA GGT CCT GGC ATC-3′;

SEQ ID NO: 9 (A-459) shows a sense DNA sequence for a siRNA or shRNA andis as follows: 5′-AAT GGC AAG ACC AGC AAG AAG-3′;

SEQ ID NO: 10 (C-454) shows a sense DNA sequence for a siRNA or shRNAcorresponding to nucleotides 454 through 474 of CyPC cDNA (Accessionnumber 571018) and is as follows: 5′-AAG ACT GAA GGT GTG CTG GTA-3′;

SEQ ID NO: 11 (NTC) shows a “no target control” DNA sequence for a siRNAor shRNA that does not have a target in the human genome and is asfollows: 5′-AAG GAG GTG ACA TCA CCA CTG-3′;

SEQ ID NO: 12 (A-forward) is a forward primer for CyPA and is asfollows: 5′-CGG GTC CTG GCA TCT TGT-3′;

SEQ ID NO: 13 (A-reverse) is a reverse primer for CyPA and is asfollows: 5′-GCA GAT GAA AAA CTG GGA ACCA-3′;

SEQ ID NO: 14 (B-forward) is a forward primer for CyPB and is asfollows: 5′-GGC CAA CGC AGG CAA A-3′;

SEQ ID NO: 15 (B-reverse) is a reverse primer for CyPB and is asfollows: 5′-TCT AGC CAG GCT GTC TTG ACT GT-3′;

SEQ ID NO: 16 (C-forward) is a forward primer for CyPC and is asfollows: 5′-GCT GAA GCA CTA TGG CAT TGG-3′;

SEQ ID NO: 17 (C-reverse) is a reverse primer for CyPC and is asfollows: 5′-GAA CTG AGA GCC ATT GGT GTC A-3′;

SEQ ID NO: 18 (IRES forward) is a forward primer for HCV internalribosome entry site (IRES) and is as follows: 5′-GTC TGC GGA ACC GGTGAG-3′;

SEQ ID NO: 19 (IRES reverse) is a reverse primer for HCV internalribosome entry site (IRES) and is as follows: 5′-CGG GTT GAT CCA AGA AAGGAC-3′;

SEQ ID NO: 20 (antisense A-285) shows an antisense RNA sequencecorresponding to a target sequence for a siRNA or shRNA and is asfollows: 5′-GAU GCC AGG ACC CGU AUG CUU-3′;

SEQ ID NO: 21 (antisense A-285′) shows an antisense RNA sequencecorresponding to a target sequence for a siRNA or shRNA and is asfollows: 5′-GAU GCC AGG ACC UGU AUG CUU-3′; and SEQ ID NO: 22 (antisenseA-459) shows an antisense RNA sequence corresponding to a targetsequence for a siRNA or shRNA and is as follows: 5′-CUU CUU GCU GGU CUUGCC AUU-3′.

DETAILED DESCRIPTION Definitions

The definition of terms departs from the commonly used meaning of theterm, applicant intends to utilize the definitions provided below,unless specifically indicated.

For the purposes of the present invention, the term “comprising” meansvarious compositions, molecules, genes, polypeptides (proteins),polynucleotides, plasmids, vectors, components, capabilities and/orsteps, etc., can be conjointly employed in embodiments of the presentinvention. Accordingly, the term “comprising” encompasses the morerestrictive terms “consisting essentially of” and “consisting of”

For the purposes of the present invention, the term “complementary” or“complementarity” refers to polynucleotides that are able to form basepairs with one another. Base pairs are typically formed by hydrogenbonds between nucleotide units in an anti-parallel orientation betweenpolynucleotide strands. Complementary polynucleotide strands can basepair in a Watson-Crick manner (e.g., A to T, A to U, C to G), or in anyother manner that allows for the formation of duplexes. As personsskilled in the art are aware, when using RNA as opposed to DNA, uracil(U) rather than thymine (T) is the base that is considered to becomplementary to adenosine. However, when a uracil is denoted in thecontext of the present invention, the ability to substitute a thymine isimplied, unless otherwise stated. “Complementarity” may exist betweentwo RNA strands, two DNA strands, or between a RNA strand and a DNAstrand. It is generally understood that two or more polynucleotides maybe “complementary” and able to form a duplex despite having less thanperfect or less than 100% complementarity. Two sequences are “perfectlycomplementary” or “100% complementary” if at least a contiguous portionof each polynucleotide sequence, comprising a region of complementarity,perfectly base pairs with the other polynucleotide without anymismatches or interruptions within such region. Two or more sequencesare considered “perfectly complementary” or “100% complementary” even ifeither or both polynucleotides contain additional non-complementarysequences as long as the contiguous region of complementarity withineach polynucleotide is able to perfectly hybridize with the other. “Lessthan perfect” complementarity refers to situations where less than allof the contiguous nucleotides within such region of complementarity areable to base pair with each other. Determining the percentage ofcomplementarity between two polynucleotide sequences is a matter ofordinary skill in the art. For purposes of RNAi, sense and antisensestrands of a siRNA or sense and antisense sequences of a shRNAcomposition may be deemed “complementary” if they have sufficientbase-pairing to form a duplex (i.e., they hybridize with each other at aphysiological temperature).

For the purposes of the present invention, the term “polynucleotide” mayrefer to either (i) a single-stranded DNA or RNA molecule or sequence,or a modified version thereof, or (ii) double-stranded DNA and/or RNAmolecules, sequences, or hybrids, or modified version(s) thereof. Forexample, the term “polynucleotide” may refer to any double-strandedpolynucleotide encoding a sense RNA sequence and an antisense RNAsequence wherein such polynucleotide sequences encoding the sense andantisense RNA sequences being directly base paired with each other.

For the purposes of the present invention, the terms “encode,”“encodes,” or “encoding” refer to the ability of an originatingpolynucleotide to be transcribed, reverse transcribed, expressed,replicated, and/or translated into the same or different polynucleotideand/or polypeptide product, as the case may be. Such polynucleotideand/or polypeptide product is described as being “encoded by” suchoriginating polynucleotide. Likewise, viral vectors may be described as“encoding” a polynucleotide or polypeptide product by containing suchoriginating polynucleotide within its genome in accordance with itsgenome type, such as single- or double-stranded DNA or RNA, as the casemay be.

For the purposes of the present invention, the term “expression vector”refers to both viral and non-viral vectors comprising a nucleic acidexpression cassette.

For the purposes of the present invention, the term “expressioncassette” is used to define a nucleotide sequence containing regulatoryelements operably linked to a coding sequence, such that the product ofthe coding sequence is expressed in a cell in a regulated or aconstitutive manner.

For the purposes of the present invention, the term “gene” refers to anucleic acid (e.g., DNA) sequence that comprises coding sequencesnecessary for the production of mRNA or polypeptide molecules or otherRNA molecules (e.g., tRNA, rRNA, etc.). However, the term gene mayfurther refer to the template strand for a gene. A functionalpolypeptide may be encoded by a full length coding sequence or by anyportion of the coding sequence of a gene so long as the desired activityor functional properties (e.g., enzymatic activity, ligand binding,signal transduction, etc.) of the full-length protein or fragment areretained. The term “gene” may also encompass sequences located adjacentto the coding region on both the 5′ and 3′ ends in addition to thecoding region of a structural gene, such that the concept of the “gene”includes other portions of the full-length mRNA, such as 5′ and 3′untranslated regions (UTRs). The term “gene” may also encompass bothcDNA and genomic sequences of a gene, and portions thereof. Such genomicsequences for a gene may further contain non-coding sequences termed“introns” or “intervening regions” or “intervening sequences,” which mayinterrupt the coding regions of a gene, termed “exons.” Such introns arespliced out of the mature mRNA sequence that is used to encode theprotein product of a gene.

For the purposes of the present invention, the term “template strand”generally refers to a DNA sequence of a gene complementary to all orapportion of a mRNA sequence expressed from such gene keeping in minddifferences between DNA and RNA sequences, such uracil in place ofthymine in RNA sequences, and vice versa.

For the purposes of the present invention, the term “coding strand”refers to a DNA sequence of a gene corresponding directly to all or aportion of a mRNA sequence expressed from such gene keeping in minddifferences between DNA and RNA sequences.

For the purposes of the present invention, the term “hepatitis C virus(HCV)” refers to the ordinary meaning of this term in the art. Thehepatitis C virus is an RNA virus of the Flaviviridae family. The terms“Hepatitis C virus” or “HCV” may include, without limitation, anypreviously identified genotype of HCV, such as genotypes 1-11 (using themost common genotyping system), which may be broken down into varioussubtypes including, without limitation, 1a, 1b, 1c, 2a, 2b, 2c, 3a, 3b,4a, 4b, 4c, 4d, 4e, 5a, 6a, 7a, 7b, 8a, 8b, 9a, 10a and 11a. Further,the terms “Hepatitis C virus” or “HCV” may include isolates taken fromindividuals comprising or consisting of closely related, yetheterogeneous, populations of viral genomes, sometimes referred to asquasi-species. In addition, the terms “Hepatitis C virus” or “HCV” mayinclude genotypes that would be understood as being HCV or sufficientlyresembling HCV to be labeled or categorized as such that have not yetemerged or been identified.

For the purposes of the present invention, the terms “homology” or“homologous” when used in the context of nucleic acid or polypeptidesequences refer to sequence identity or similarity between two or moresequences. The degree of sequence identity is generally quantified usingpercentages, which is calculated based on the number of differingnucleotides or amino acids over the total length of the sequence.Determining the percentage of homology is a matter of skill in the art.For example, sequence alignment programs may be used to aid thedetermination of the degree of identity or homology between two or moresequences. Homologous sequences may comprise two or more DNA sequences,two or more RNA sequences, or at least one DNA sequence and at least oneRNA sequence. When comparing DNA and RNA sequences, it is generallyunderstood that a uracil (U) in a RNA sequence is considered equivalentto a thymine (T) in a DNA sequence for purposes of determining homology.

For the purposes of the present invention, the term “small interferingRNA” or “siRNA” refers to double-stranded RNA molecules, comprising asense strand and an antisense strand, having sufficient complementarityto one another to form a duplex. Such sense and antisense strands eachhave a region of complementarity ranging, for example, from about 10 toabout 30 contiguous nucleotides that base pair sufficiently to form aduplex or double-stranded siRNA. Such siRNAs are able to specificallyinterfere with the expression of a gene by triggering the RNAi machineryof a cell to remove RNA transcripts having identical or homologoussequences to the siRNA sequence. As described herein, the sense andantisense strands of siRNA may each consist of only complementaryregions, or one or both strands may comprise additional sequences,including non-complementary sequences, such 5′ and 3′ overhangs. Inaddition, such siRNAs may have other modifications, such as, forexample, substituted or engineered nucleotides or other sequences, whichcontribute to either the stability of the siRNA, its delivery to a cellor tissue, or its potency in triggering RNAi. It is to be understoodthat the terms “strand” and “oligonucleotide” may be usedinterchangeably in reference to the sense and antisense strands of siRNAcompositions.

For the purposes of the present invention, the terms “patient,”“subject,” or “individual” as used herein may be used interchangeablyand generally refer to any animal, preferably mammals, that may beinfected or at risk of infection by HCV. Such terms may refer to anyanimal having domesticated, agricultural, commercial, or other researchuses. The terms “patient,” “subject,” or “individual” may also refer tohumans.

For the purposes of the present invention, the term “3′ overhang” refersto at least one unpaired nucleotide extending out from the 3′-end of atleast one strand of the duplexed siRNA and may engineered into an shRNA.Similarly, the term “5′ overhang” refers to at least one unpairednucleotide extending out from the 5′-end of at least one strand of theduplexed siRNA.

For the purposes of the present invention, the term “region” whenapplied to polynucleotides generally refers to a contiguous portion orsequence of a single-stranded or double-stranded polynucleotidemolecule(s). However, the term “region” may also refer to an entiresingle-stranded or double-stranded polynucleotide molecule(s).

For the purposes of the present invention, the term “physiologicalconditions” refers to conditions that approximate the chemical and/ortemperature environment that may exist within the body of an individual,subject, or patient.

For the purposes of the present invention, the term “physiologicaltemperature” generally refers to a temperature present within the bodyof an individual, subject, or patient. The term “physiologicaltemperature” may be assumed to be approximately 37° C. unless otherwisespecified.

For the purposes of the present invention, the term “sense RNA” refersto a RNA sequence corresponding to all or a portion of a coding sequenceof a gene or all or a portion of a plus (+) strand or mRNA sequencegenerated from a gene, or a RNA sequence homologous thereto, keeping inmind the differences between RNA and DNA molecules.

For the purposes of the present invention, the term “antisense RNA”refers to a RNA sequence corresponding to all or a portion of a templatesequence of a gene, or a sequence homologous thereto, or a minus (−)strand or all or a portion of a sequence complementary to a mRNAsequence generated from a gene, keeping in mind the differences betweenRNA and DNA molecules.

For the purposes of the present invention, the term “hybridize” refersto associating two complementary nucleic acid strands to form adouble-stranded molecule which may contain two DNA strands, two RNAstrands, one DNA and one RNA strand, etc.

Relative to U.S. Provisional App. No. 60/948,040, sequence ranges andnucleotide numbers within CyPA cDNA have been updated herein to moreconsistently match cDNA sequences as provided by accession number Y00052and SEQ ID NO: 1. For example, the nucleotide sequence range 157 to 185referred to in U.S. Provisional App. No. 60/948,040 has beencorrespondingly updated herein to nucleotides 155 to 183, nucleotidesequence range 161 to 181 referred to in U.S. Provisional App. No.60/948,040 has been correspondingly updated herein to nucleotides 159 to179, and A161 and sh-A161 referred to in U.S. Provisional App. No.60/948,040 have been correspondingly renamed as A159 and sh-A159,respectively. Unless otherwise stated, these sequence ranges andnucleotide numbers referred to herein are the updated or renamed rangesand numbers.

Description

RNAi, as originally discovered in invertebrates and employed dsRNAs withlength greater than 30 nucleotides, is not effective in mammalian cells.This was found to be due to the fact that long dsRNAs (greater than 30nucleotides) elicit interferon responses, resulting in nonspecific mRNAdegradation and inhibition of protein synthesis. This problem may beovercome by using smaller double-stranded siRNAs, e.g. 20-23 nucleotidesin length for each strand, which do not induce an interferon responseyet remain potent and specific inhibitors of endogenous gene expression.See, e.g., Elbashir, S. M. et al., “RNA interference is mediated by 21-and 22-nucleotide RNAs,” Genes Dev. 15:188 (2001).

In research laboratories, two types of siRNA (or shRNA) have been widelyused to suppress exogenous as well as endogenous gene expression:synthetic siRNA and vector-based siRNA (i.e., in vivo transcribedsiRNA). Synthetic siRNAs are generally synthesized in vitro prior toadministration to cells; however, vector-based siRNAs are expressed fromvectors introduced into cells. The vector-based approach is oftencarried out using short or small hairpin RNAs (shRNAs). Vector-basedapproaches may use RNA polymerase III promoters, such as H1 promoter andU6 promoter to drive transcription of a shRNA. The shRNA transcript mayconsist of a 19- to 29-bp RNA stem, with the two complementary (senseand antisense) strands joined by a tightly structured hairpin or loop.The shRNA may be processed in the cell into siRNA through the action ofthe Dicer family of enzymes. Thus, the transcribed products may mimicthe synthetic siRNA duplexes and may be as effective as the syntheticsiRNA for suppressing their corresponding genes.

Previously, the most effective HCV therapy employed a combination ofalpha-interferon and ribavirin, leading to sustained efficacy in only40% of patients. In addition, recent clinical results have also shownthat combined interferon and cyclosporine A treatment may be moreeffective in treating patients with chronic hepatitis C than interferonmonotherapy, which may suggest a role for cyclosporin A as a validtreatment option for HCV. See, e.g., Inoue, K. et al., “Combinedinterferon a2b and cyclosporine A in the treatment of chronic hepatitisC: controlled trial,” Journal of Gastroenterology 38(6):567-72 (2003).

However, treatment of HCV infection has been met with less thansatisfactory results primarily because of the resistance to interferon-a(IFN) and possible side effects of existing therapies. Most currentefforts to develop new HCV drugs have focused on viral targets, such asinhibitors of viral protease and polymerase enzymes. However, resistanceto these inhibitors readily develops in vitro and in vivo. New classesof safe and broadly-acting treatments are therefore urgently needed.

siRNAs targeting HCV are known. However, these siRNAs directly targetthe viral genome, which mutates rapidly. Thus, resistance to such siRNAsquickly develops when mutations at siRNA target sites arise. Therefore,a need for the development of novel compositions and methods for thetreatment, maintenance, inhibition, prevention, etc., of HCV infectioncontinues.

Cyclophilins (CyPs) are a family of cellular enzymes possessing thepeptidyl-prolyl isomerase activity. The prototypical member of the CyPfamily is Cyclophilin A (CyPA), the main intracellular ligand ofcyclosporine (CsA) (see, e.g., Handschumacher, R. E. et al.,“Cyclophilin: a specific cytosolic binding protein for cyclosporine A,”Science 226:544-547 (1984)), an immunosuppressant which is usually usedto suppress rejection after internal organ transplants. CyPA exists inthe cytosol and has a beta barrel structure with two alpha helices and abeta sheet. CyPA plays the role of a molecular chaperone, i.e., it helpsother proteins fold correctly.

The role of human CyPs as cellular cofactors in HCV replication issuggested by studies that show that CsA is effective in suppressing HCVreplication. See, e.g., Nakagawa, M., N. et al. Specific inhibition ofhepatitis C virus replication by cyclosporin A,” Biochem. Biophys. Res.Commun. 313:42-47 (2004); Watashi, K. et al., “Cyclosporin A suppressesreplication of hepatitis C virus genome in cultured hepatocytes,”Hepatology 38:1282-1288 (2003). Subsequently, a correlation between theCyP-binding and anti-HCV activity has been observed for derivatives ofCsA. See, e.g., Ma, S. et al., “NIM811, a cyclophilin inhibitor,exhibits potent in vitro activity against hepatitis C virus alone or incombination with alpha interferon,” Antimicrob. Agents Chemother.50:2976-2982 (2006); Watashi, K. et al., “Cyclophilin B is a functionalregulator of hepatitis C virus RNA polymerase,” Mol. Cell. 19:111-122(2005). But despite both protein binding and resistance mapping studiesthat might suggest that NS5B is a viral target for CsA (see, e.g.,Fernandes, F. D. et al., “Sensitivity of hepatitis C virus tocyclosporine A depends on nonstructural proteins NS5A and NS5B,”Hepatology 46:1026-1033 (2007); Robida, J. M. et al., “Characterizationof hepatitis C virus subgenomic replicon resistance to cyclosporine invitro” J. Virol. 81:5829-5840 (2007); Watashi, K. et al., “Cyclophilin Bis a functional regulator of hepatitis C virus RNA polymerase,” Mol.Cell. 19:111-122 (2005)), the identities and relative contributions ofthe various CyPs implicated in this interaction remain debatable (see,e.g., Nakagawa, M. et al., “Suppression of hepatitis C virus replicationby cyclosporine a is mediated by blockade of cyclophilins,”Gastroenterology 129:1031-1041 (2005); Robida, J. M. et al.,“Characterization of hepatitis C virus subgenomic replicon resistance tocyclosporine in vitro. J. Virol. 81:5829-5840 (2007); Watashi, K. et al.Cyclophilin B is a functional regulator of hepatitis C virus RNApolymerase. Mol. Cell. 19:111-122 (2005)).

As described herein, it has been found that CyPA, and not CyPB or CyPC,is an essential cofactor for the replication of various HCV isolates andgenotypes. Therefore, according to some embodiments of the presentinvention, compositions and methods are provided for the treatmentand/or prevention of HCV infection in a cell or an individual, subject,or patient using an inhibitor of CyPA. More particularly, suchcompositions and methods may comprise siRNAs, shRNAs, or otherRNAi-mediated compositions that specifically target CyPA to effectivelyinhibit, prevent, treat, and/or manage HCV infection and/or disease.Applicants describe herein that siRNA or shRNA compositions of thepresent invention that target a specific region of CyPA corresponding toat least 19 contiguous nucleotides from about nucleotide 155 to aboutnucleotide 183 of the coding sequence of human CyPA cDNA (accessionnumber Y00052; SEQ ID NO: 1) are especially potent and effective attargeting and inhibiting CyPA as well as the production of HCV virions.

It has been further found herein that CyPA is the principal mediator ofCsA resistance in vitro and that Cyclosporin A-resistant (CsA-resistant)HCV strains show less dependency on CyPA. However, depletion of CyPAfrom host cells by RNAi has also been found herein to sensitizeCsA-resistant HCV to CsA treatment. According to some embodiments,siRNA, shRNA, or other RNAi-mediated compositions and methods accordingto embodiments of the present invention may specifically target CyPA andmay be further used in combination with other drugs or therapies, suchas interferon and/or CsA. Such combined therapies may have cumulative orsynergistic effects in treating, managing, inhibiting, preventing, etc.,HCV infection.

In general, siRNA molecules which are directed against viral targets,including Hepatitis C virus, may not provide a long-term cure orprotection against the virus due to their ability of viruses, includingHCV, to mutate quickly, and therefore avoid recognition by siRNAs. Incontrast, the embodiments of the present invention may provide siRNA orshRNA compositions that specifically target a cellular gene, i.e., CyPA,thus encoding a protein necessary for reproduction of HCV in host cells.Since such a target gene is present within the host cell genome, it maynot be subject to rapid mutational changes that occur with many viraltargets.

Among the various embodiments of the present invention is a smallinterfering RNA (siRNA) comprising a sense RNA sequence and an antisenseRNA sequence which form an RNA duplex, wherein the sense RNA sequence isat least about 70% homologous to at least 19 contiguous nucleotides fromnucleotide 155 to nucleotide 183 of human cyclophilin A cDNA sequence(with accession number Y00052; SEQ ID NO: 1) and wherein the antisenseRNA sequence is complementary thereto. However, the degree of homologybetween sense RNA sequence of siRNA and at least 19 contiguousnucleotides of CyPA between nucleotides 155 and 183 of the codingsequence of CyPA cDNA may be higher, such as, for example, at leastabout 80%, 90%, 95%, or 100% homologous or identical, with antisense RNAsequence complementary thereto.

Described a different way, the antisense RNA sequence of siRNA ofembodiments of the present invention may be at least about 70%complementary to at least 19 contiguous nucleotides between nucleotide155 and nucleotide 183 of the coding sequence of human cyclophilin AcDNA (accession number Y00052) with sense RNA sequence complementary tosuch antisense RNA sequence or able to hybridize to the sense RNAsequence under physiological conditions. However, the degree ofcomplementarity between antisense strand of siRNA and at least 19contiguous nucleotides of CyPA between nucleotides 155 and 183 of thecoding sequence of CyPA cDNA may be higher, such as, for example, atleast about 80%, 90%, 95%, or 100% complementary, with a sense RNAsequence complementary thereto or able to hybridize thereto.

Alternatively, the sense and antisense RNA strands of siRNAs may beexpressed as a single transcript, such that the sense and antisense RNAare covalently linked by a hairpin or stem-loop sequence to form ashRNA. Such shRNA may be cleaved when introduced into or expressed by acell to yield two separate strands similar to the siRNA.

Embodiments of compositions of the present invention may further includeany DNA polynucleotide sequences that encode any of the embodiments ofthe sense and/or antisense RNA sequences or strands of the presentinvention as described herein. Such DNA sequences may be placed intovectors, such as plasmids, viral vectors, etc., to achieve expression ofthe desired siRNA or shRNA. Expression from such vectors may becontrolled using promoters known or used in the art.

According to some embodiments of the present invention, siRNA and shRNAcompositions may be formulated as a pharmaceutical composition with apharmaceutically acceptable carrier or in combination with deliveryagents known in the art. Such siRNA and/or shRNA compositions maycomprise two or more siRNAs and/or shRNAs having distinct sequences.Furthermore, such siRNA and/or shRNAs compositions may be used incombination with other known therapies or drugs.

In other embodiments of the present invention, methods for inhibitinghepatitis C virus replication and/or infection are provided, whereinsuch methods comprise introducing into a cell or tissue an siRNA orshRNA composition according to embodiments of the present invention (asdescribed herein) that targets CyPA to inhibit HCV infection. Suchmethods for inhibiting HCV may be used to treat, manage, inhibit,prevent, etc., HCV infection in an individual, subject, or patientinfected or at risk of infection by HCV and may be performed incombination with other therapies or drugs.

siRNA and shRNA Characteristics

According to some embodiments of the present invention, a smallinterfering RNA (siRNA) composition is provided, comprising a sense RNAsequence and an antisense RNA sequence corresponding to a region ofCyPA, which has been shown to be involved in HCV infection. Thus, byintroducing such siRNA composition into a cell, the level, expression,and/or activity of CyPA is reduced via RNAi and HCV infection isinhibited. According to one set of embodiments, for example, a sense RNAsequence (or strand) of siRNA of the present invention is at least about70% homologous to at least 19 contiguous nucleotides between nucleotide155 and nucleotide 183 of the coding sequence of human cyclophilin AcDNA (with accession number Y00052; SEQ ID NO: 1) with antisense RNAsequence (or strand) complementary to such sense RNA sequence (i.e.,able to hybridize to the sense RNA sequence at a physiologicaltemperature). However, the degree of homology between sense RNA sequenceof siRNA and the at least 19 contiguous nucleotides of CyPA betweennucleotides 155 and 183 of the coding sequence of CyPA cDNA may behigher, such as, for example, at least about 80%, 90%, 95%, or 100%homologous or identical, with antisense RNA sequence complementarythereto. According to some embodiments, sense RNA strand of siRNAcomposition may comprise SEQ ID NO: 2 corresponding to nucleotides 159through 179 of CyPA, with an antisense strand complementary thereto.

According to some embodiments of the present invention, the antisenseRNA sequence of siRNA is at least about 70% complementary to at least 19contiguous nucleotides between nucleotide 155 and nucleotide 183 of thecoding sequence of human cyclophilin A cDNA (with accession numberY00052; SEQ ID NO: 1) with sense RNA sequence complementary to suchantisense RNA sequence (i.e., able to hybridize to the sense RNAsequence at a physiological temperature). However, the degree ofcomplementarity between an antisense strand of siRNA and at least 19contiguous nucleotides of CyPA between nucleotides 155 and 183 of thecoding sequence of CyPA cDNA may be higher, such as, for example, atleast about 80%, 90%, 95%, or 100% complementary, with sense RNAsequence complementary thereto.

Other embodiments of compositions of the present invention may furtherinclude any DNA polynucleotide sequences that encode any of the senseand/or antisense siRNA sequences or strands or shRNA sequences accordingto embodiments of the present invention as described herein. Asdescribed in greater detail below, such DNA sequences may be placed intovectors, such as plasmids, viral vectors, etc., to achieve expression ofthe desired siRNA or shRNA. The sense and antisense RNA sequences may beexpressed by the same vector or separately by different vectors. Forexample, where the sense and antisense RNA sequences are expressed bythe same vector, two promoters may be placed in opposite orientationsflanking the portion of the DNA polynucleotide encoding the sense RNAsequence and the antisense RNA sequence from opposing strands that aredirectly base-paired with one another. Alternatively, for example, thesense and antisense RNA sequences may be expressed from a DNApolynucleotide as a single RNA molecule, such as a shRNA, linked by ahairpin sequence inserted between them. Therefore, embodiments of thepresent invention may further include any DNA polynucleotide sequencethat encodes a shRNA sequence having a sense RNA sequence and anantisense RNA sequence covalently linked by a hairpin RNA sequence. Forexample, DNA sequence encoding shRNA may comprise SEQ ID NO: 5.

The sense and antisense sequences of siRNA compositions according toembodiments of the present invention are at least partiallycomplementary to each other to form a duplex that is sufficient totrigger RNAi-mediated degradation of its target. However, it is to beunderstood that the degree of homology between the sense and antisensestrand sequences of the siRNA and the coding and template sequences ofhuman cyclophilin A, respectively, may be different (i.e., the degree ofcomplementarity between the sense and antisense strands of siRNA of thepresent invention may be less than 100% and still have sufficientbase-pairing to form a duplex and elicit specific RNAi-mediateddestruction of its target, such as CyPA). One skilled in the art mayachieve a desired homology and/or complementarity by makingsubstitutions in either or both of the sense and antisense strandsequences as discussed below. It should be noted, however, thatoff-targeting and non-specific binding of sense or antisense RNAsequences should be avoided as much as possible so that ideally only thetarget, such as CyPA, is noticeably affected. Therefore, the actualdegree of freedom available to relax the amount of homology betweensense sequence of siRNA or shRNA of present invention and its targetsequence may be limited. It should also be noted that the degree ofcomplementarity between sense and antisense strands of siRNA should besufficient to form a duplex such that RNAi-mediated degradation of itstarget is triggered.

The region of human CyPA corresponding to nucleotides 155 through 183 ofthe coding sequence of human cyclophilin A cDNA is specificallyidentified and chosen herein due to its potency in downregulating humanCyPA and inhibiting HCV infection in cells as described herein. However,it is to be understood that embodiments of siRNA and shRNA compositionsof the present invention may conceivably include other previouslyunknown contiguous sequences of human CyPA, including sequences outsideof nucleotides 155 through 183, which may be effective at reducing thelevel, expression, activity, etc., of CyPA and/or HCV infection whenintroduced into cells. Furthermore, embodiments of siRNA or shRNAcompositions of the present invention may comprise combinations of twoor more siRNAs and/or shRNAs including those according to embodiments ofthe present invention having distinct sequences.

As mentioned above, human cyclophilin A sequence is known and availablethrough GenBank (accession number Y00052). One skilled in the art mayreadily determine the proper nucleotide numbering for CyPA, such as theregion comprising nucleotides 155 through 183 of the human cyclophilin AcDNA sequence (SEQ ID NO: 1). However, the genomic or cDNA sequence ofhuman cyclophilin A may also be identified by different accessionnumbers, clones, or other identifiers. Embodiments of siRNA or shRNAcompositions of the present invention may include any siRNA or shRNAsequences homologous to a region of a cyclophilin A gene or homologuederived from any non-human animal species that may experience HCVinfection. For example, a region corresponding to nucleotides 155through 183 of the human cyclophilin A cDNA sequence may be identifiedand used. A person skilled in the art would know how to find previouslyidentified CyPA gene or homologue sequences from other species throughstandard sequence searching techniques.

One skilled in the art may be able to determine the appropriate numberand type of substitutions or modifications in either the sense orantisense siRNA or shRNA strands or sequences relative to coding andtemplate sequences of target, respectively, such that sufficienthomology and complementarity is afforded to maintain base-pairing andduplex formation between sense and antisense strands or sequences tocause RNAi-mediated degradation of a target, such as CyPA. When makingany such substitutions, considerations such as where they are introducedand whether they are dispersed throughout the sequence or occur togethermay affect the efficacy of the siRNA. By way of example, it is known inthe art that substitutions in the center of the molecule tend to affectthe efficacy to a greater degree than the substitutions at either end ofthe molecule. Similarly, two or more contiguous substitutions tend toaffect binding or hybridization of sense and antisense strands to agreater extent than two or more spaced-apart mutations. Accordingly,substitutions may be introduced such that there are regions of at least3 contiguous unmutated nucleotides between each substitution. Forexample, there may be at least 4 unmutated contiguous nucleotides, e.g.,at least 5 unmutated contiguous nucleotides between each substitution.

The following features are not required, but may be useful, whendetermining which substitutions might be desirable in choosing siRNA orshRNA sequences that improve efficacy for RNAi-mediated compositionsaccording to embodiments of the present invention: (1) a G/C content ofabout 25% to about 60% G/C; (2) at least 3 A/Us at positions 15-19 ofthe sense strand; (3) no internal repeats; (4) an A at position 19 ofthe sense strand; (5) an A at position 3 of the sense strand; (6) a U atposition 10 of the sense strand; (7) no G/C at position 19 of the sensestrand; and (8) no G at position 13 of the sense strand.

In addition, if a substitution might result in a siRNA or shRNA sequencehaving one or more of the following criteria, such sequence may be lesslikely to function successfully as a siRNA or shRNA: (1) a sequencecomprising a stretch of 4 or more of the same base in a row; (2) asequence comprising homopolymers of Gs; (3) a sequence comprising triplebase motifs (e.g., GGG, CCC, AAA, or TTT); (4) a sequence comprisingstretches of 7 or more G/Cs in a row; and (5) a sequence comprisingdirect repeats of 4 or more bases within the candidates resulting ininternal fold-back structures. However, such sequences may still beevaluated for the ability to function as siRNA or shRNA molecules. Forfurther discussion of design guidelines that may be useful in designingsiRNA or shRNA sequences for compositions and methods according toembodiments of the present invention directed against CyPA, see, e.g.,De Paula, D. et al., “Hydrophobization and bioconjugation for enhancedsiRNA delivery and targeting. RNA,” 13: 431-56 (2007); and Chen, Y. etal., “RNAi for Treating Hepatitis B Viral Infection” PharmaceuticalResearch 25(1): 72-86 (2008), the entire disclosures and contents ofwhich are hereby incorporated by reference.

Accordingly, one skilled in the art may be able to determine whichsubstitutions might be appropriate in designing siRNA or shRNAcompositions according to embodiments of the present invention thatwould more likely target CyPA effectively. Based on known rules andprinciples, a person skilled may be able to determine appropriatemodifications to the sense and/or antisense strands of a CyPA siRNA orshRNA according to embodiments of the present invention, such thatsufficient complementarity and/or homology is more likely maintained toelicit RNAi-mediated degradation of CyPA despite having less than 100%homology with CyPA and/or less than 100% complementarity between senseand antisense strands of the siRNA.

Furthermore, siRNA or shRNA polynucleotides may be chemicallysynthesized or modified, e.g., for purposes of reducingimmunostimulatory effect of siRNA sequences, affecting the potency ofRNAi, increasing stability of siRNA or shRNA molecules, targeting tospecific cells or tissues, and/or strengthening hybridization betweensense and antisense strands. This may employ any known nucleotidederivatives or methods known or available in the art. Knowledge relatingto backbone modifications used with antisense ODNs may be readilyadapted to develop new siRNA or shRNA technologies. For example, thechemically modified siRNA may comprise modified nucleotides including,but not limited to, 2′-O-Me nucleotides, 2′-O-allyl nucleotides,2′-deoxy-2′-fluoro (2′-F) nucleotides, 2′ deoxy nucleotides,2′-O-(2-methoxyethyl) (MOE) nucleotides, locked nucleic acid (LNA)nucleotides, phosphorothioate (PS) linkages, and combinations thereof.In some preferred embodiments, the modified siRNA may comprise 2′-O-Mepurine and/or pyrimidine nucleotides, such as, for example,2′-O-Me-guanosine nucleotides, 2′-O-Me-uridine nucleotides,2′-O-Me-adenosine nucleotides, 2′-O-Me-cytosine nucleotides, andmixtures thereof. Either or both sense and/or antisense sequences orstrands of siRNA or shRNA according to embodiments of present inventionmay comprise one or more synthesized or modified nucleotides.

In addition, siRNA compositions may further contain bioconjugates whichmay be useful to: (1) further increase their thermodynamic and nucleasestability; (2) improve the biodistribution and pharmacokinetic profilesof siRNAs; and/or (3) target them to specific cell types. For furtherdiscussion of available chemical modifications and bioconjugates thatmay be used in combination with embodiments of compositions and methodsof the present invention, see, e.g., De Paula, D. et al.,“Hydrophobization and bioconjugation for enhanced siRNA delivery andtargeting,” RNA 13:431-56 (2007); Chen, Y. et al., “RNAi for TreatingHepatitis B Viral Infection,” Pharmaceutical Research 25(1):72-86(2008); and Kim, D. et al., “RNAi mechanisms and applications,”Biotechniques 44(5):613-16 (2008), the entire disclosures and contentsof which are hereby incorporated by reference.

According to some embodiments, the sense RNA strand or sequence of thesiRNA or shRNA directed against cyclophilin A is from about 19 to about29 nucleotides long. For example, embodiments of the sense strand of thepresent invention may be from 20 to 28 nucleotides long, such as from 21to 25 nucleotides long. Therefore, these sense strands of siRNAmolecules may be 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29nucleotides in length. Similarly, the antisense strand of embodiments ofthe siRNA of the present invention may also be from 19 to 29 nucleotideslong, for example, from 20 to 28 nucleotides long, such as from 21 to 25nucleotides long. Therefore, these antisense strands of siRNA moleculesmay be 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides inlength. According to some embodiments, such lengths correspond exactlyto the length of a region of complementarity between sense and antisenseRNA sequences of siRNA or shRNA. Such regions of complementarity presentwithin sense and antisense strands or sequences may have the samelength. However, such sense and antisense strands may further containnon-complementary sequences, such as 3′ overhangs (see below).

The at least 19 contiguous nucleotides of both sense RNA sequence andantisense RNA sequence of siRNA or shRNA may directly hybridize to oneanother along most if not all of such length. According to someembodiments of the present invention, the sense and antisense strands orsequences of the siRNA or shRNA composition may only contain sequencesthat are complementary to the other strand or sequence. However,according to other embodiments, the sense and antisense strands orsequences of the siRNA or shRNA composition may further contain othernon-complementary sequences that may provide different functions for thesiRNA or shRNA composition that do not contribute to base-pairingbetween the sense and antisense strands or sequences.

According to some embodiments, the sense and antisense strands of thesiRNA may comprise two complementary, single-stranded RNA molecules.According to other embodiments, however, the sense RNA sequence and theantisense RNA sequence may be encoded by a single molecule with the twocomplementary sequences (corresponding to sense and antisense strands)covalently linked by a single-stranded “hairpin” or loop sequence.Without being bound by any theory, it is believed that the hairpinsequence of the latter type of the shRNA is cleaved intracellularly bythe “Dicer” protein (or its equivalent) to form an siRNA or equivalentthat may then be used to achieve RNAi-mediated degradation of a target(See, e.g., Tuschl, T., “Expanding small RNA interference,” Nat.Biotechnol, 20(5):446-448 (2002)). The hairpin sequence may be fromabout 4 to about 12 nucleotides in length. For example, the hairpinsequence may be 9 nucleotides in length.

As an example of an embodiment of a shRNA of the present invention, SEQID NO: 3 provides a sequence having a sense RNA sequence and anantisense RNA sequence covalently attached by a hairpin sequence.However, such shRNA merely provides an example of a sense and antisensesequence attached by a hairpin sequence. Any acceptable hairpin sequencemay be used to covalently link any sense sequence with any antisensesequence of the present invention as described herein. Indeed,embodiments of the present invention may comprise any sense sequence andantisense sequence described herein attached by any acceptable hairpinsequence, such as 5′-UUCAAGAGA-3′. However, it is to be understood thatsince a hairpin sequence does not necessarily correspond to a sequencewithin a targeted gene, such as CyPA, any acceptable hairpin sequencemay be used as a part of embodiments for shRNAs of the presentinvention.

The embodiments of siRNA or shRNA compositions of the present inventionmay comprise partially purified RNA, substantially pure RNA, syntheticRNA, or recombinantly produced RNA, as well as altered RNA that differsfrom naturally-occurring RNA by the addition, deletion, substitution,synthesis, and/or modification of one or more nucleotides. Suchmodifications may include addition of non-nucleotide material, such asto the end(s) of the siRNA or to one or more internal nucleotides of thesiRNA, including modifications that make the siRNA or shRNA moreeffective or resistant to nuclease digestion.

In addition to complementary sequences, one or both strands ofembodiments of the siRNA or shRNA compositions of the present inventionmay further comprise a 5′ overhang and/or a 3′ overhang. According tosome embodiments, either or both strands of the siRNA may comprise a 3′overhang of from 1 to about 6 nucleotides (which may include eitherribonucleotides or deoxyribonucleotides) in length, for example, from 1to about 5 nucleotides in length, including from 1 to about 4nucleotides in length, such as 2 to 4 nucleotides in length. Accordingto those embodiments in which both strands of the siRNA comprise a 3′overhang, the length of the overhangs may be the same or different foreach strand. According to some embodiments, the 3′ overhang present oneither or both strands of the siRNA may be 2 nucleotides in length. Forexample, each strand of the siRNA of the invention may comprise 3′overhangs of dithymidylic acid (“TT”) or diuridylic acid (“UU”) or othereffective dinucleotide combination known in the art. The 5′ end of oneor both strands or sequences of a siRNA or shRNA may also contain aphosphate group.

In order to enhance the stability of the present siRNA or shRNA, the 3′overhangs may be also stabilized against degradation. For example, theoverhangs may be stabilized by including purine nucleotides, such asadenosine or guanosine nucleotides. Alternatively, substitution ofpyrimidine nucleotides by modified analogues, e.g., substitution ofuridine nucleotides in the 3′ overhangs with 2′-deoxythymidine, may betolerated and not affect the efficiency of RNAi degradation. Inparticular, the absence of a 2′-hydroxyl in the 2′-deoxythymidine maysignificantly enhance the nuclease resistance of the 3′ overhang intissue culture medium. Despite being made predominantly ofribonucleotides, it is also possible that embodiments of the siRNAs orshRNAs of the present invention may be synthesized or modified tocontain one or more deoxyribonucleotides in addition to ribonucleotidesalong the length of one or both strands or sequences to improve efficacyor stability as the case may be.

siRNA Preparation

Embodiments of siRNA or shRNA compositions may be prepared in a numberof ways, such as by chemical synthesis, in vitro transcription ordigestion, or by endogenous expression. For further discussion ofmethods of siRNA production or synthesis, see, e.g., De Paula, D. etal., “Hydrophobization and bioconjugation for enhanced siRNA deliveryand targeting” RNA 13:431-56 (2007); Chen, Y. et al., “RNAi for TreatingHepatitis B Viral Infection,” Pharmaceutical Research 25(1):72-86(2008); and Kim, D. et al., “RNAi mechanisms and applications,”Biotechniques 44(5):613-16 (2008), the entire disclosures and contentsof which are hereby incorporated by reference.

By way of example, in vitro transcription may be performed with a T7polymerase, and siRNA digestion may be carried out by treating longdouble stranded RNA (dsRNA) prepared by one of the two previous methodswith Dicer enzyme. Dicer enzyme may create mixed populations of dsRNAfrom about 21 to about 23 base pairs in length from dsRNA that is fromabout 500 base pairs to about 1000 base pairs in size. Dicer enzyme mayeffectively cleave modified strands of dsRNA, such as 2′ fluoro-modifieddsRNA. The Dicer enzyme method of preparing embodiments of siRNAs of thepresent invention may be performed using a Dicer siRNA Generation Kitavailable from Gene Therapy Systems (San Diego, Calif.).

In one embodiment, the siRNA may be synthetically produced. By way ofexample and not of limitation, embodiments of the siRNAs of the presentinvention may be chemically synthesized using appropriately protectedribonucleotides phosphoramidites with a conventional DNA/RNAsynthesizer. The siRNA may be synthesized as two separate, complementaryRNA molecules, or as a single RNA molecule with two complementaryregions. Commercial suppliers of synthetic RNA molecules or synthesisreagents include Proligo (Hamburg, Germany), Dharmacon Research(Lafayette, Colo. USA), Pierce Chemical (part of Perbio Science,Rockford, Ill., USA), Glen Research (Sterling, Va. USA), ChemGenes(Ashland, Mass. USA) and Cruachem (Glasgow, UK).

The embodiments of siRNA of the present invention may also berecombinantly produced in vitro using plasmid vectors. A variety ofdifferent vectors may be employed for producing siRNAs by recombinanttechniques. Such vectors are well known in the art and may include,e.g., chromosomal or nonchromosomal vectors, derivatives of SV40,bacterial plasmids, phage, baculovirus, yeast plasmids, vectors derivedfrom combinations of plasmids and phage DNA, viral DNA such as vaccinia,adenovirus, fowl pox virus, and pseudorabies. However, any vector may beused as long as it is replicable and viable in a desired host forexpression. The embodiments of siRNA of the present invention may beexpressed from a recombinant plasmid either as two separate,complementary RNA molecules, or as a single RNA molecule with twocomplementary regions, such as a shRNA.

Plasmids suitable for expressing embodiments of siRNAs or shRNAs of thepresent invention, methods for inserting nucleic acid sequencesexpressing or encoding siRNAs or shRNAs into a plasmid, and methods fordelivering recombinant plasmids to cells of interest are known in theart. See, for example, Tuschl, T., “Expanding small RNA interference,”Nat. Biotechnol. 20(5):446-448 (2002); Brummelkamp, T. R. et al., “Asystem for stable expression of short interfering RNAs in mammaliancells,” Science 296(5567):550-553 (2002); Miyagishi M et al., “U6promoter-driven siRNAs with four uridine 3′ overhangs efficientlysuppress targeted gene expression in mammalian cells,” Nat. Biotechnol.20(5):497-500 (2002); Paddison, P. J. et al., “Short hairpin RNAs(shRNAs) induce sequence-specific silencing in mammalian cells,” GenesDev. 16(8):948-58 (2002); Lee, N. S. et al., “Expression of smallinterfering RNAs targeted against HIV-1 rev transcripts in human cells,”Nat. Biotechnol. 20(5):500-505 (2002); Paul, C. P. et al., “Effectiveexpression of small interfering RNA in human cells,” Nat. Biotechnol.20(5): 505-508 (2002); De Paula, D. et al., “Hydrophobization andbioconjugation for enhanced siRNA delivery and targeting,” RNA 13:431-56 (2007); Chen, Y. et al., “RNAi for Treating Hepatitis B ViralInfection,” Pharmaceutical Research 25(1):72-86 (2008); and Kim, D. etal., “RNAi mechanisms and applications,” Biotechniques 44(5):613-16(2008), the entire disclosures and contents of which are herebyincorporated by reference.

siRNA or shRNA molecules may also be endogenously produced in cellsbeing tested. For example, extra-chromosomal plasmids or vectors may beintroduced into cells via electroporation, microinjection, or complexformation with synthetic carriers (such as lipids, polymers, orpeptides). Such plasmids or vectors may be transiently or stablyexpressed by cells. Alternatively, siRNA or shRNA molecules may also beproduced from viral vectors. Viral vectors suitable for use in thepresent invention are well known in the art. See, for example, Dornburg,R., “Reticuloendotheliosis viruses and derived vectors,” Gene Therap.2:301-310 (1995); Eglitis, M. A., “Retroviral vectors for introductionof genes into mammalian cells,” Biotechniques 6(7):608-614 (1988);Miller A D, Hum Gene Therap. 1:5-14 (1990); Anderson, W. F., “Human genetherapy,” Nature 392(6679 Suppl.):25-30 (1998); De Paula, D. et al.,“Hydrophobization and bioconjugation for enhanced siRNA delivery andtargeting. RNA,” 13:431-56 (2007); Chen, Y. et al., “RNAi for TreatingHepatitis B Viral Infection,” Pharmaceutical Research 25(1):72-86(2008); and Kim, D. et al., “RNAi mechanisms and applications”Biotechniques 44(5):613-16 (2008), the entire disclosures and contentsof which are hereby incorporated by reference.

Appropriate DNA segments may be inserted into a vector by a variety ofprocedures. In general, DNA sequences may be inserted into anappropriate restriction endonuclease site(s) by procedures known in theart, which may be performed without undue experimentation by a skilledartisan. A DNA segment in an expression vector may be operatively linkedto an appropriate expression control sequence(s) (i.e., a promoter) todirect siRNA or shRNA synthesis. Such promoters may include any promoterknown in the art for expression either in vivo or in vitro. Suitableeukaryotic promoters may include, e.g., CMV immediate early promoter,the herpes simplex virus (HSV) thymidine kinase promoter, the early andlate SV40 promoters, the promoters of retroviral long terminal repeats(LTRs), such as those of the Rous Sarcoma Virus (RSV), andmetallothionein promoters, such as the mouse metallothionein-I promoter.The promoters used in embodiments of the present invention may includeRNA polymerase III promoters. For example, the promoters may be selectedfrom the group consisting of the U6, H1, 5S, 7SK, and tRNA promoters,e.g., the promoter is a U6 or a H1 promoter.

The promoters which may be used in embodiments of the present inventionmay also be inducible, such that expression may be turned “on” or “off”For example, a tetracycline-regulatable system employing the U6 promotermay be used to control the production of siRNA. Additionally, promoterswhich are tissue specific or respond to a particular stimulus may alsobe used. By way of example and not of limitation, tissue specificpromoters include promoters which are active in the liver, such as,e.g., albumin promoter. Promoters which respond to a particular stimulusmay include, e.g., heat shock protein promoters, and Tet-off and Tet-onpromoters.

In addition, the expression vectors may contain one or more selectablemarker genes, such as dihydrofolate reductase or neomycin resistance foreukaryotic cell culture, to allow selection of transformed host cells orcells being treated. In addition, one or more selectable markers may beused to allow selection in prokaryotic cells, such as tetracycline orampicillin resistance.

According to some embodiments of the present invention, a vector may beprovided comprising a DNA segment encoding a sense RNA strand operablylinked to a first promoter and an antisense (opposite) RNA strandoperably linked to a second promoter. According to this embodiment, eachRNA strand or sequence may be independently expressed, and the promoterdriving expression of each strand may be the same or different from theother promoter used to express the other strand or sequence. In otherembodiments, the vector used to express each strand of an embodiment ofsiRNA of the present invention may include opposing promoters. Forexample, the vector may contain two promoters, such as a T7 or U6promoter, on either side of a DNA segment encoding each strand of thesiRNA and placed in opposing orientations, with or without atranscription terminator placed between the two opposing promoters.See., e.g., Wang, Z. et al., “Inhibition of Trypanosoma brucei GeneExpression by RNA Interference Using an Integratable Vector withOpposing T7 Promoters,” J. Biol. Chem. 275:40174-40179 (2000), theentire disclosure and contents of which is hereby incorporated byreference.

According to other embodiments, the DNA segments encoding both siRNAstrands are under the control of a single promoter. According to someother embodiments, the DNA segment encoding each complementary strand(i.e., sense and antisense strands) may contain a hairpin or loop regioninterspersed or inserted between the two complementary strands orsequences, such that transcription yields one RNA transcript with bothcomplementary sequences covalently linked by a hairpin or loop (whichmay be referred to as “shRNA”). The single transcript may, in turn,anneal to itself creating a “hairpin” RNA structure capable of inducingRNAi. The hairpin or loop may be from about 4 to about 12 nucleotides inlength, such as 9 nucleotides in length.

As an embodiment of an shRNA of the present invention, SEQ ID NO: 3 mayprovide a sequence having a sense RNA sequence and an antisense RNAsequence covalently attached by a hairpin sequence. However, such shRNAmerely provides an example of a sense and antisense sequence attached bya hairpin sequence. Any acceptable hairpin sequence may be used tocovalently link any sense sequence with any antisense sequence accordingto embodiments of the present invention as described herein. Forexample, embodiments of the present invention may comprise any sensesequence and antisense sequence described herein attached by anyacceptable hairpin sequence, such as 5′-UUCAAGAGA-3′. It is to beunderstood that since a hairpin sequence does not necessarily correspondto a sequence within a targeted gene, such as CyPA, any acceptablehairpin sequence may be used as part of shRNA embodiments of the presentinvention.

Detection of siRNA Inhibition

The ability of a particular siRNA or shRNA composition to causeRNAi-mediated degradation of a target mRNA, reduce expression of atarget protein, and/or inhibit virus infection and reproduction in cellsmay be evaluated using standard techniques. For example, any of themethods described herein may be used. In the recent years, developmentof cell culture systems permissible for HCV replication and infectionhas greatly aided in vitro study of HCV as described in, e.g., Lohmannet al., “Replication of subgenomic hepatitis C virus in a hepatoma cellline. Science,” 285:110-113 (1999); Blight et al., “Efficient initiationof HCV RNA replication in cell culture,” Science 290:1972-1974 (2000);Wakita, T. et al., “Production of hepatitis C virus in tissue culturefrom a cloned viral genome,” Nature Medicine 11(7):791-6 (2005);Lindenbach, B. D. et al., “Complete replication of hepatitis C virus incell culture,” Science 309:623-626 (2005); and Zhong, J. et al., “Robusthepatitis C virus infection in vitro” PNAS 102(26):9294-9 (2005), theentire disclosures and contents of which are hereby incorporated byreference.

For example, Wakita et al. developed an HCV genotype 2a replicon (JFH-1)that replicates efficiently in Huh-7 cells, other humanhepatocyte-derived cells (e.g., HepG2 and IMY-N9), and nonhepatic cells(e.g., HeLa and HEK293) without adaptive mutations but with lowerinfection efficiency. This JFH-1 genome also turned out to be capable ofproducing infectious viruses in cell culture. Using part of the JFH-1genome, Lindenbach et al. (supra) described a full-length HCV genomethat replicated and produced virus particles that are infectious in cellculture (HCVcc). Replication of HCVcc is robust, producing nearly 10⁵infectious units per milliliter within 48 hours. Virus particles arefilterable and neutralized with a monoclonal antibody against the viralglycoprotein E2.

As another example, Zhong et al. also developed a robust in vitroinfection system based on Huh-7-derived cell lines and the JFH-1consensus clone. This system yields viral titers of 10⁴-10⁵ infectiousunits per ml of culture supernatant. In addition, infection spreadsthroughout the culture within a few days after inoculation at lowmultiplicities of infection (moi), and the virus can be seriallypassaged without loss in infectivity.

Thus, in order to test the efficacy of a siRNA or shRNA to prevent ortreat HCV replication and/or infection, the following exemplary generalprotocol may be used. Briefly, HCV RNA may be delivered to cells byelectroporation or liposome-mediated transfection. For purposes ofelectroporation, trypsinized cells that are to be transformed with HCVRNA are washed twice with and resuspended in serum-free Opti-MEM(Invitrogen) at 1×10⁷ cells per ml. Ten micrograms of HCV RNA is mixedwith 0.4 ml of the cells in a 4-mm cuvette, and a Bio-Rad Gene Pulsersystem is used to deliver a single pulse at 0.27 kV, 100 ohms, and 960μF. The cells are then plated in a T162 Costar flask (Corning). Liposomemediated transfection may be performed with Lipofectamin 2000(Invitrogen) at an RNA/lipofectamin ratio of 1:2 by using 5 μg of HCVRNA in cell suspensions containing 10⁴ cells. Cells are plated in DMEMwith 20% FCS for overnight incubation. Whether HCV RNA is introduced byelectroporation or liposome-mediated, transfected cells are transferredto complete DMEM and cultured for at least two days. Cells are passagedevery 3-5 days. The presence of HCV in these cells and correspondingsupernatants may be determined before transfection of siRNA or shRNAinto the cells, and at desired time points after transfection of thecells with siRNA or shRNA. The siRNA or shRNA compositions, or DNAcompositions or vectors encoding such siRNAs or shRNAs, may beintroduced into HCV positive cells by any known method including thosedescribed in the present application. However, the above example ismerely exemplary. One skilled in the art may readily performmodifications to the above protocol or use entirely different methodsknown in the art.

Total cellular RNA may be isolated by the guanidine thiocyanate methodby using standard protocols, and RT-QPCR analysis may be performed asdescribed in, e.g., Kapadia, S. B. et al., “Hepatitis C virus RNAreplication is regulated by host geranylgeranylation and fatty acids”PNAS 102:2561-2566 (2005). For RT-QPCR, HCV and GAPDH transcript levelsmay be determined relative to a standard curve comprised of serialdilutions of plasmid containing the HCV cDNA or human GAPDH gene.According to one example for titration of HCV, cell supernatants may be,e.g., serially diluted 10-fold in complete DMEM and used to infect 10⁴naive Huh-7.5.1 or Huh-7 cells per well in 96-well plates (Corning).According to this example, the inoculum is incubated with cells for 1 hat 37° C. and then supplemented with fresh complete DMEM. The level ofHCV infection may then be determined, e.g., 3 days post-infection byimmunofluorescence staining for HCV NS5A. The viral titer is expressedas focus-forming units per milliliter of supernatant (ffu/ml),determined by the average number of NS5A-positive foci detected at thehighest dilutions. HCV RNA levels and the number of NS5A-positive fociobtained before and after the introduction of siRNA into cells may beused to determine the efficacy of siRNA in treating HCV. Additionalmethods that are well known in the art for determining the levels of HCVin cells before and after the siRNA treatment may also be used.

RNAi-mediated degradation of target mRNA by a siRNA or shRNA containinga given target sequence may also be evaluated using animal models forHCV infection. In one embodiment, chimpanzees are used. By way ofexample, HCV RNA has been delivered to chimpanzees by at least oneintrahepatic injection. See, e.g., Kolykhalov et al., “Transmission ofhepatitis C by intrahepatic inoculation with transcribed RNA,” Science277:570-574 (1997). According to this example, serum samples arecollected, e.g., every 2 weeks to test for HCV replication. Briefly,tests that may be done include assays for liver enzymes, antibodies toHCV and viremia, as determined by quantifying circulating HCV RNA bybranched DNA and quantitative-competitive (QC) RT-PCR. After a desirabletime period, e.g., two months, HCV positive chimpanzees may beadministered embodiments of a siRNA or shRNA of the present invention,such as by infection with lentiviral vectors encoding such siRNA orshRNA. The siRNA or shRNA compositions may be delivered once or as manytimes as necessary to control the infection. Serum samples are againcollected at predetermined time periods, e.g., every two weeks todetermine the levels of HCV replication by the same tests as describedabove. The efficacy of a particular siRNA or shRNA may be established bycomparing the results of the tests performed before and after siRNAadministration. For example, stabilization of levels of liver enzymesand/or lower numbers of circulating HCV may be used to indicate thatsiRNA is effective at reducing HCV replication and/or infection.

Another animal model that may be used is a chimeric scid/Alb-uPA mouse,which expresses urokinase-type plasminogen activator under the controlof the albumin promoter. Since the expression of this transgene is toxicto mouse hepatocytes, these mice either die early due to a bleedingphenotype or at 2-3 weeks of age due to liver failure. See, e.g., Heckelet al., “Neonatal bleeding in transgenic mice expressing urokinase-typeplasminogen activator,” Cell 62:447-456 (1990). However, these mice maybe transplanted with human hepatocytes, resulting in repopulation ofmouse liver. Once these mice are transplanted to achieve chimeric liver,they may be used as a model for HCV infection. Briefly, once infectedwith HCV, these mice may be treated with a siRNA or shRNA of the presentinvention to determine its efficacy against HCV. The same types ofassays as described above for the chimpanzee model may be used.Furthermore, mice may be sacrificed and their livers examinedhistologically for HCV infection and/or tissue damage.

As another example, cells from an individual, subject, or patientinfected with HCV may be treated with embodiments of a siRNA or shRNAcomposition of the present invention in vitro and tested forRNAi-mediated degradation of target mRNA. Methods for isolating andculturing cells from a patient are well known in the art.

siRNA Delivery

In embodiments of the methods of the present invention, the siRNA ofshRNA may be administered to an individual, subject, or patient eitheras a naked siRNAs and/or shRNAs or as part of a recombinant plasmid orvector expressing such siRNAs and/or shRNAs, which may also be deliveredin conjunction with a delivery reagent. Alternatively, embodiments ofthe siRNA or shRNA compositions of the present invention may beadministered as a viral vector(s) encoding either separate sense andantisense siRNA strands or a single shRNA. When naked siRNAs, shRNAs, orrecombinant plasmids or vectors expressing a siRNA or shRNA areadministered directly to cells, such delivery may be achieved, forexample, by electroporation, gene gun, microinjection, or complexformation with synthetic carriers (such as lipids, polymers, and/orpeptides). In addition, embodiments of the siRNAs or shRNAs of thepresent invention may be delivered as a pharmaceutical composition incombination with a pharmaceutically acceptable carrier.

Suitable delivery reagents for administration in conjunction with thesiRNA or shRNA may include cationic polymers and lipids as well asencapsulated lipid particles, such as liposomes, etc. Examples mayinclude Mirus Transit TKO lipophilic reagent, lipofectin, lipofectamine,cellfectin, or polycations (e.g., polylysine), or liposomes. For furtherdiscussion of effective delivery reagents that may be used incombination with present siRNA compositions, see, e.g., De Paula, D. etal. “Hydrophobization and bioconjugation for enhanced siRNA delivery andtargeting,” RNA 13:431-56 (2007); Chen, Y. et al., “RNAi for TreatingHepatitis B Viral Infection,” Pharmaceutical Research 25(1):72-86(2008); and Kim, D. et al., “RNAi mechanisms and applications,”Biotechniques 44(5):613-16 (2008), the entire disclosures and contentsof which are hereby incorporated by reference. See also, e.g., U.S.patent application Ser. Nos. 11/598,052, 11/978,398, 11/978,455,11/978,457, 11/746,864, 11/750,553, and 10/597,431, the entiredisclosures and contents of which are hereby incorporated by reference.

According to some embodiments, the delivery reagent may be a liposome.Liposomes may be used to aid in the delivery of the siRNA or shRNA to aparticular tissue, such as the liver, and can also increase the bloodhalf-life of the siRNA. The selection of lipids is generally guided byconsideration of factors such as the desired liposome size and half-lifeof the liposomes in the blood stream. Methods for the preparation ofliposomes as delivery agents that may be used in combination withembodiments of compositions and methods of the present invention arewell known in the art. For example, see Szoka et al., “Comparativeproperties and methods of preparation of lipid vesicles (liposomes),”Ann. Rev. Biophys. Bioeng. 9:467 (1980); Immordino, M. L., “Stealthliposomes: review of the basic science, rationale, and clinicalapplications, existing and potential,” Int. J. Nanomedicine 1(3):297-315(2006); Samad, A, “Liposomal Drug Delivery Systems: An Update Review,”Current Drug Delivery 4(4):297-305 (2007); and U.S. Pat. Nos. 4,235,871,4,501,728, 4,837,028, and 5,019,369, the entire disclosures and contentsof which are hereby incorporated by reference. The liposomesencapsulating the siRNA may comprise a ligand molecule that targets theliposome to the liver. Such ligands may include, but are not limited to,ligand of the asialoglycoprotein receptor (ASGPR),β-sitosterol-β-d-glucoside (sito-G), galactosylated liposomes,asialofetuin grafted vesicles, liposomes that are modified with humanserum albumin, and mannosylated liposomes. See, e.g., Chen, Y. et al.,“RNAi for Treating Hepatitis B Viral Infection,” Pharmaceutical Research25(1):72-86 (2008); and Pathak, A., “Nanovectors for efficient liverspecific gene transfer,” Int. J. Nanomedicine 3(1):31-49 (2008), theentire disclosures and contents of which are hereby incorporated byreference.

The liposomes encapsulating the siRNA may also be modified so as toavoid clearance by the mononuclear macrophage and reticuloendothelialsystems, for example by having opsonization-inhibition moieties bound tothe surface of the structure. In one embodiment an embodiment of aliposome of the present invention may comprise bothopsonization-inhibition moieties and a ligand. Opsonization-inhibitingmoieties for use in preparing the embodiment of the liposomes of thepresent invention may be large hydrophilic polymers that are bound tothe liposome membrane. As used herein, an opsonization inhibiting moietyis “bound” to a liposome membrane when it is chemically or physicallyattached to the membrane, e.g., by the intercalation of a lipid-solubleanchor into the membrane itself, or by binding directly to active groupsof membrane lipids. These opsonization-inhibiting hydrophilic polymersform a protective surface layer which significantly decreases the uptakeof the liposomes by the macrophage-monocyte system (“MMS”) andreticuloendothelial system (“RES”); e.g., as described in U.S. Pat. No.4,920,016. Liposomes modified with opsonization-inhibition moieties thusremain in the circulation much longer than unmodified liposomes and aresometimes called “stealth” liposomes.

Opsonization-inhibiting moieties suitable for modifying liposomes aregenerally water-soluble polymers with a number-average molecular weightfrom about 500 to about 40,000 daltons, and more preferably from about2,000 to about 20,000 daltons. Such polymers may include polyethyleneglycol (PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxyPEG or PPG, and PEG or PPG stearate synthetic polymers such aspolyacrylamide or poly N-vinyl pyrrolidone; linear, branched, ordendrimeric polyamidoamines; polyacrylic acids; polyalcohols, e.g.,polyvinylalchohol and polyxylitol to which carboxylic or amino groupsare chemically linked, as well as gangliosides, such as ganglioside GM₁.Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof,may also be suitable. In addition, the opsonization inhibiting polymermay be a block copolymer of PEG and either a polyamino acid,polysaccharide, polyamidoamine, polyethyleneamine, or polynucleotide.The opsonization-inhibiting polymers may also be natural polysaccharidescontaining amino acids or carboxylic acids, e.g., galacturonic acid,glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid,neuraminic acid, alginic, acid, carrageenan; aminated polysaccharides oroligosaccharides (linear or branched); or carboxylated polysaccharidesor oligosaccharides, e.g., reacted with derivatives of carbonic acidswith resultant linking of carboxylic groups. For example, theopsonization-inhibiting moiety may be a PEG, PPG, or derivativesthereof. Liposomes modified with PEG or PEG-derivatives are sometimescalled “PEGylated liposomes.”

The opsonization-inhibiting moiety may be bound to the liposome membraneby any one of numerous well-known techniques. For example, anN-hydroxysuccinimide ester of PEG may be bound to aphosphatidyl-ethanolamine lipid-soluble anchor and then bound to amembrane. Similarly, a dextran polymer may be derivatized with astearylamine lipid-soluble anchor via reductive amination usingNa(CN)BH3 and a solvent mixture such as tetrahydrofuran and water in a30:12 ratio at 60° C.

The vector containing the appropriate DNA sequence as described herein,as well as an appropriate promoter or control sequence, may be employedto transform an appropriate host to permit the host to express the siRNAor shRNA. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts have been described and are well knownin the art. See, e.g., Sambrook, et al., Molecular Cloning: A LaboratoryManual, (Second Edition, Cold Spring Harbor, N.Y. 1989). In oneembodiment, the cells used to produce the siRNAs or shRNAs may be HEK293T cells.

As described above, embodiments of the siRNA or shRNA compositions ofthe present invention may also be delivered using viral vectors bymodifying methods generally known in the art. Examples of viral vectorsthat may be suitable for use with embodiments of methods of the presentinvention may include retroviral, adenoviral, and adeno-associated viralvectors. Any viral vector capable of encoding or accepting codingsequences for a siRNA or shRNA molecule(s) to be expressed may be usedincluding, for example, vectors derived from adenovirus (AV),adeno-associated virus (AAV), retroviruses (e.g., lentiviruses),Rhabdoviruses, herpes virus, etc. According to some preferredembodiments, viral vectors that may be used to deliver embodiments ofsiRNA compositions of the present invention may include lentiviruses orlentiviral-derived vectors. The tropism of the viral vectors may also bemodified by pseudotyping the vectors with envelope proteins or othersurface antigens from other viruses. For example, an AAV vector of theinvention may be pseudotyped with surface proteins from vesicularstomatitis virus (VSV), rabies, Ebola, Marburg, and the like. Accordingto some embodiments, siRNAs or shRNA may be expressed using RNApolymerase III promoters, such as U6, H1, or tRNA promoters.

For further review and discussion of viral vectors that may potentiallybe used in combination with the present invention, see, e.g., De Paula,D. et al., “Hydrophobization and bioconjugation for enhanced siRNAdelivery and targeting,” RNA 13:431-56 (2007); Chen, Y. et al., “RNAifor Treating Hepatitis B Viral Infection,” Pharmaceutical Research25(1):72-86 (2008); and Kim, D. et al., “RNAi mechanisms andapplications,” Biotechniques 44(5):613-16 (2008), the entire disclosuresand contents of which are hereby incorporated by reference.

Suitable enteral administration routes for administering embodiments ofsiRNA or shRNA compositions of the present invention include oral,rectal or intranasal delivery. Suitable parenteral administration routesmay include intravascular administration (e.g., intravenous bolusinjection, intravenous infusion, intra-arterial bolus injection,intra-arterial infusion and catheter instillation into the vasculature);peri- and intra-tissue administration (e.g., intra-hepatic injection),subcutaneous injection or deposition including subcutaneous infusion(such as by osmotic pumps), direct application to the area at or near asite of infection or risk of infection (e.g., by a catheter or otherplacement device), and inhalation. In an embodiment, injections orinfusions of the siRNA or shRNA are given directly into the liver oradjacent thereto. See, e.g., Chen, Y. et al., “RNAi for TreatingHepatitis B Viral Infection,” Pharmaceutical Research 25(1):72-86(2008), the entire disclosure and contents of which is herebyincorporated by reference.

The above-mentioned general methods for siRNA or shRNA delivery to cellsmay be further exemplified without limitation by the following specificexamples of gene therapy techniques for mammalian liver. For example,cultured hepatocytes have been genetically modified by retroviralvectors (Wolff, J. A. et al., PNAS 84:3344-3348 (1987); Ledley, F. D. etal., PNAS 84:5335-5339 (1987)) and re-implanted back into the livers inanimals and in people (J. R. Chowdhury et al., Science 254:1802 (1991);Grossman, M. et al., Nature Genetics 6:335 (1994)). Retroviral vectorshave also been delivered directly to livers in which hepatocyte divisionis induced by partial hepatectomy (Kay, M. A. et al., Hum Gene Ther.3:641-647 (1992); Ferry, N. et al., PNAS 88:8377-8381 (1991); Kaleko, M.et al., Hum Gene Ther. 2:27-32 (1991). The injection of adenoviralvectors into the portal or systemic circulatory systems led to highlevels of foreign gene expression that is transient(Stratford-Perricaudet, L. D. et al., Hum. Gene Ther. 1:241 (1990);Jaffe, H. A. et al., Nat. Genet. 1:372 (1992); Li, Q. et al., Hum. GeneTher. 4:403 (1993). Non-viral transfer methods may include polylysinecomplexes of asialoglycoproteins that are injected into the systemcirculation (Wu, G. Y. et al., Biol. Chem. 263:14621-14624 (1988)).Foreign gene expression has also been achieved by repetitively injectingnaked DNA in isotonic solutions into the liver parenchyma of animalstreated with dexamethasone (Malone, R. W. et al., JBC 269:29903-29907(1994); Hickman, M. A., Human Gene Ther. 5:1477-1483 (1994). Plasmid DNAexpression in the liver has also been achieved via liposomes deliveredby tail vein or intraportal routes (Kaneda, Y. et al., Biol. Chem.264:12126-12129 (1989); Soriano, P. et al., PNAS 80:7128-7131 (1983);Kaneda, Y., et al., Science 243:375-378 (1989). However, it is to beunderstood that any appropriate siRNA or shRNA delivery method that iscurrently known or available in the art may be used in combination withor as part of embodiments of compositions and methods of the presentinvention. For example, transposon-mediated vectors may be used todeliver siRNA or shRNA compositions of the present invention to cells.

Embodiments of compositions of the present invention used in combinationwith delivery reagents may comprise an siRNA comprising a sense RNAsequence and an antisense RNA sequence, wherein sense RNA sequence ofsiRNA may be at least about 70% homologous to at least 19 contiguousnucleotides between nucleotide 155 and nucleotide 183 of the codingsequence of human cyclophilin A cDNA (accession number Y00052; SEQ IDNO: 1) with antisense RNA sequence complementary to such sense RNAsequence (i.e., able to hybridize to the sense RNA sequence at aphysiological temperature). However, the degree of homology betweensense strand of siRNA and the at least 19 contiguous nucleotides of CyPAbetween nucleotides 155 and 183 of the coding sequence of CyPA cDNA maybe higher, such as, for example, at least about 80%, 90%, 95%, or 100%homologous or identical, with antisense strand complementary thereto.According to one embodiment, sense strand of siRNA composition maycomprise SEQ ID NO: 2 corresponding to nucleotides 159 through 179 ofCyPA cDNA along with an antisense strand complementary thereto.

Described a different way, such delivery embodiments of compositions ofthe present invention may comprise a siRNA comprising a sense RNAsequence and an antisense RNA sequence, wherein the antisense RNAsequence of the embodiment of siRNA of the present invention may be atleast about 70% complementary to at least 19 contiguous nucleotidesbetween nucleotide 155 and nucleotide 183 of the coding sequence ofhuman cyclophilin A cDNA (accession number Y00052; SEQ ID NO: 1) withsense RNA sequence complementary to such antisense RNA sequence (i.e.,able to hybridize to the sense RNA sequence at a physiologicaltemperature). However, the degree of complementarity between antisensestrand of siRNA and the at least 19 contiguous nucleotides of CyPAbetween nucleotides 155 and 183 of the coding sequence of CyPA cDNA maybe higher, such as, for example, at least about 80%, 90%, 95%, or 100%complementary, with a sense RNA sequence complementary thereto. As anexample, antisense sequence may comprise SEQ ID NO: 4.

As described above, such sense and antisense RNA sequences ofembodiments of the present invention may be alternatively expressed as asingle molecule covalently attached by a hairpin sequence to form ashRNA. In addition, embodiments of the present invention may include DNApolynucleotides encoding or expressing such siRNA or shRNA molecules,and such DNA polynucleotides may be constructed as part of an expressionvector or plasmid. Therefore, delivery embodiments of compositions ofthe present invention may further include DNA polynucleotides, vectors,plasmids, and shRNAs in combination with a suitable delivery reagent.Such delivery reagents may contain two or more such siRNA, shRNA, DNApolynucleotide, vector, and/or plasmid compositions together or withother known drugs or therapies.

Pharmaceutical Compositions

The embodiments of siRNA, shRNA, or DNA compositions of the presentinvention may be formulated as a pharmaceutical composition incombination with a pharmaceutically acceptable carrier according totechniques known in the art. Embodiments of pharmaceutical compositionsof the present invention may be characterized as being sterile andpyrogen-free. Methods for preparing embodiments of pharmaceuticalcompositions of the present invention are well within the skill in theart, for example as described in Remington's Pharmaceutical Science,(17th ed., Mack Publishing Company, Easton, Pa., 1985); Goodman &Gillman's: The Pharmacological Basis of Therapeutics (11th Edition,McGraw-Hill Professional, 2005); and Griffin P. et al. The Textbook ofPharmaceutical Medicine (Blackwell Publishing, Malden, Mass., 2006), theentire disclosures and contents of which are hereby incorporated byreference.

The present pharmaceutical formulations may comprise a siRNA or shRNA(e.g., 0.1 to 90% by weight), or a physiologically acceptable saltthereof, mixed with a pharmaceutically acceptable carrier.Physiologically acceptable carriers may include water, buffered water,saline solutions (e.g., normal saline or balanced saline solutions suchas Hank's or Earle's balanced salt solutions), 0.4% saline, 0.3%glycine, hyaluronic acid, etc.

Such embodiments of pharmaceutical compositions of the present inventionmay comprise an siRNA comprising a sense RNA sequence and an antisenseRNA sequence, wherein sense RNA sequence of siRNA may be at least about70% homologous to at least 19 contiguous nucleotides between nucleotide155 and nucleotide 183 of the coding sequence of human cyclophilin AcDNA (accession number Y00052; SEQ ID NO: 1) with antisense RNA sequencecomplementary to such sense RNA sequence. However, the degree ofhomology between sense strand of siRNA and the at least 19 contiguousnucleotides of CyPA between nucleotides 155 and 183 of the codingsequence of CyPA cDNA may be higher, such as, for example, at leastabout 80%, 90%, 95%, or 100% homologous or identical, with antisensestrand complementary thereto. According to one embodiment, the sensestrand of siRNA composition may comprise SEQ ID NO: 2 corresponding tonucleotides 159 through 179 of CyPA cDNA along with an antisense strandcomplementary thereto.

Described a different way, such pharmaceutical compositions may comprisean siRNA comprising a sense RNA sequence and an antisense RNA sequence,wherein antisense RNA sequence of siRNA of the present invention may beat least about 70% complementary to at least 19 contiguous nucleotidesbetween nucleotide 155 and nucleotide 183 of the coding sequence ofhuman cyclophilin A cDNA (accession number Y00052; SEQ ID NO: 1) withsense RNA sequence complementary to such antisense RNA sequence.However, the degree of complementarity between antisense strand of siRNAand at least 19 contiguous nucleotides of CyPA between nucleotides 155and 183 of the coding sequence of CyPA cDNA may be higher, such as, forexample, at least about 80%, 90%, 95%, or 100% complementary, with asense RNA sequence complementary thereto. As an example, the antisensesequence may comprise SEQ ID NO: 4.

Again, as described above, such sense and antisense RNA sequences ofembodiments of the present invention may be alternatively expressed as asingle molecule covalently attached by a hairpin sequence to form ashRNA. In addition, embodiments of the present invention include DNApolynucleotides encoding or expressing such siRNA or shRNA molecules,and such DNA polynucleotides may be constructed as part of an expressionvector or plasmid. Therefore, embodiments of pharmaceutical compositionsof the present invention may further include DNA polynucleotides,vectors, plasmids, and/or shRNAs in combination with an acceptablecarrier. In addition, pharmaceutical compositions may further comprisetwo or more different embodiments of siRNA, shRNA, DNA polynucleotide,vector, and/or plasmid compositions of the present invention, togetheror in combination with other known drugs or therapies.

Embodiments of pharmaceutical compositions of the present invention maybe administered orally, nasally, parenterally, intrasystemically,intraperitoneally, topically (as by drops or transdermal patch),bucally, intrahepatically, or as an oral or nasal spray.

An embodiment of a pharmaceutical composition of the present inventionfor parenteral injection may comprise pharmaceutically acceptablesterile aqueous or nonaqueous solutions, dispersions, suspensions oremulsions as well as sterile powders for reconstitution into sterileinjectable solutions or dispersions just prior to use. Examples ofsuitable aqueous and nonaqueous carriers, diluents, solvents or vehiclesinclude water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, etc.), carboxymethylcellulose and suitable mixturesthereof, vegetable oils (such as olive oil), and injectable organicesters such as ethyl oleate. Proper fluidity may be maintained, forexample, by the use of coating materials such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants.

Injectable depot forms may be made by forming microencapsule matrices ofthe siRNA, shRNA, etc., in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of drug to polymerand the nature of the particular polymer employed, the rate of releasemay be controlled. Examples of other biodegradable polymers may includepoly(orthoesters) and poly(anhydrides). Depot injectable formulationsmay also prepared by entrapping the siRNA, shRNA, etc., composition inliposomes or microemulsions which are compatible with body tissues.

The injectable formulations may be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium just prior to use.

In some cases, to prolong the effect of siRNAs, shRNAs, etc., it isdesirable to slow the absorption from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the composition may then depend upon its rate ofdissolution which, in turn, may depend upon crystal size and crystallineform. Alternatively, delayed absorption of a parenterally administeredform may be accomplished by dissolving or suspending the drug in an oilvehicle. Prolonged absorption of the injectable pharmaceuticalcomposition may be brought about by the inclusion of agents which delayabsorption such as aluminum monostearate and gelatin.

Solid dosage forms for oral administration may include, but are notlimited to, capsules, tablets, pills, powders, and granules. In suchsolid dosage forms, the composition of the present invention may bemixed with at least one pharmaceutically acceptable excipient orcarrier, such as sodium citrate or dicalcium phosphate and/or: (a)fillers or extenders such as starches, lactose, sucrose, glucose,mannitol, and silicic acid; (b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone,sucrose, and acacia; (c) humectants such as glycerol; (d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate; (e) solutionretarding agents such as paraffin; (f) absorption accelerators such asquaternary ammonium compounds; (g) wetting agents such as, for example,acetyl alcohol and glycerol monostearate; (h) absorbents such as kaolinand bentonite clay; and/or (i) lubricants such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, and mixtures thereof. In the case of capsules, tablets andpills, the dosage form can also comprise buffering agents. Soft and hardfilled gelatin capsules may also be used excipients, such as lactose ormilk sugar as well as high molecular weight polyethylene glycols and thelike.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules may be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and may also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which may beused may include polymeric substances and waxes.

The embodiments of the pharmaceutical compositions of the presentinvention may also be in micro-encapsulated form, if appropriate, withone or more of the above-mentioned excipients.

Liquid dosage forms for oral administration may include, but are notlimited to, pharmaceutically acceptable emulsions, solutions,suspensions, syrups and elixirs. Liquid dosage forms may contain inertdiluents commonly used in the art such as, for example, water or othersolvents, solubilizing agents and/or emulsifiers such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ,olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof. Besides inert diluents, oral compositions may also includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, or perfuming agents. Suspensions may containsuspending agents, such as ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, and/ortragacanth, and mixtures thereof.

Alternatively, the composition may be pressurized and contain acompressed gas, such as nitrogen or a liquefied gas propellant. Theliquefied propellant medium and indeed the total composition maypreferably be such that the composition does not dissolve therein to anysubstantial extent. The pressurized composition may also contain asurface active agent. The surface active agent may be a liquid or solidnonionic surface active agent or may be a solid anionic surface activeagent. It would be generally preferred that the solid anionic surfaceactive agent be in the form of a sodium salt.

Embodiments of the pharmaceutical compositions of the present inventionmay also comprise conventional pharmaceutical excipients and/oradditives. Suitable pharmaceutical excipients may include stabilizers,antioxidants, osmolality adjusting agents, buffers, and/or pH adjustingagents. Suitable additives include physiologically biocompatible buffers(e.g., tromethamine hydrochloride), additions of chelants (such as, forexample, DTPA or DTPA-bisamide) or calcium chelate complexes (as forexample calcium DTPA, CaNaDTPA-bisamide), and/or, optional additions ofcalcium or sodium salts (for example, calcium chloride, calciumascorbate, calcium gluconate or calcium lactate).

Pharmaceutical compositions comprising an embodiment of a siRNA or shRNAof the present invention may include penetration enhancers to enhancetheir delivery, such as through the alimentary route. Penetrationenhancers may be classified as belonging to one of five broadcategories, i.e., fatty acids, bile salts, chelating agents, surfactantsand non-surfactants (Lee et al., Critical Reviews in Therapeutic DrugCarrier System, 8:91-192 (1991); Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 7:1-33 (1990)). One or morepenetration enhancers from one or more of these broad categories may beincluded. Various fatty acids and their derivatives which act aspenetration enhancers may include, for example, oleic acid, lauric acid,capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid,linolenic acid, dicaprate, tricaprate, recinleate, monoolein (a.k.a.1-monooleoyl-glycerol), dilaurin, caprylic acid, arachidonic acid,glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, mono- and di-glycerides and physiologically acceptablesalts thereof (i.e., oleate, laurate, caprate, myristate, palmitate,stearate, linoleate, etc.). See, e.g., Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, page 92 (1991); Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 7:1 (1990); E1-Hariri etal., The mitigating effects of phosphatidylcholines on bile salt- andlysophosphatidylcholine-induced membrane damage,” J. Pharm. Pharmacol.44:651-654 (1992)).

Chelating agents may include, but are not limited to, disodiumethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g.,sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines) (Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, page 92 (1991); Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 7:1 (1990); Buur et al., J. ControlRel., 14:43-51 (1990)). Chelating agents may have the added advantage ofalso serving as DNase inhibitors.

Surfactants may include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, page 92(1991)); and perfluorochemical emulsions, such as FC43 (Takahashi etal., J. Pharm. Phamacol., 40:252-257 1988)).

Non-surfactants may include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, page 92 (1991));and non-steroidal anti-inflammatory agents such as diclofenac sodium,indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol.,39:621-626 1987)).

Prevention of degradation of siRNAs, shRNAs, etc., by microorganisms maybe ensured by the inclusion of various antibacterial and antifungalagents including, for example, paraben, chlorobutanol, phenol, sorbicacid, etc.

One skilled in the art will appreciate that an effective amount ofsiRNA, shRNA, etc. of the present invention may be determinedempirically and may be employed in pure form or, where such forms exist,in a pharmaceutically acceptable salt, ester or prodrug form. A“therapeutically effective” amount of an embodiment of a siRNA, shRNA,or siRNA/shRNA-expressing vector composition of the present inventionmay be determined by the amount needed to treat, manage, inhibit,prevent or ameliorate adverse conditions or symptoms of HCV infection ordisease. Such determination may be made according to any method known inthe art or described herein, such as, e.g., by measuring the level,amount, and/or activity of HCV particles and various liver enzymes or bymonitoring symptoms of infection or disease. Embodiments of compositionsof the present invention may be administered to an individual, subjector patient, in need of treatment for HCV infection or at risk of HCVinfection, either directly to a cell or tissue, as a pharmaceuticalcomposition, or in combination with a delivery reagent as describedabove. It will be understood that, when administering compositions ofthe present invention to a human patient, a “therapeutically effective”amount is expressed as total daily usage of the embodiment of thecomposition of the present invention and may be decided by the attendingphysician within the scope of sound medical judgment. The specific“therapeutically effective” dose level for a particular individual,subject, or patient may depend upon a variety of factors, including, forexample, the type and degree of the cellular or physiological responsedesired, activity of the specific siRNA or shRNA composition employed orexpressed, the specific pharmaceutical formation or delivery methodemployed, the age, body weight, general health, sex and diet of thepatient, the time of administration, route of administration, and rateof excretion of the composition in relation to HCV infection, thespecific dosage regimen, drugs used in combination or coincidental withthe present composition, and other factors well known in the medicalarts. For example, doses of embodiments of compositions of the presentcompositions may be started at levels lower than those expected to benecessary to achieve the desired therapeutic effect and to graduallyincrease the dosages until the desired effect is achieved.

One skilled in the art may also readily determine an appropriate dosageregimen for administering the embodiments of the compositions of thepresent invention to a given individual, subject, or patient. Forexample, a siRNA, shRNA or vector composition may be administered to asubject once, such as by a single injection or deposition.Alternatively, a siRNA, shRNA or vector composition may be administeredto a subject multiple times daily or weekly. For example, compositionsmay be administered to a subject once weekly for a period of from aboutthree to about twenty-eight weeks, such as from about seven to about tenweeks. In an example of a dosage regimen, compositions may be injectedat or near the liver once a week for seven weeks.

In another embodiment, embodiments of compositions of the presentinvention may be administered in combination with other agents known orused to treat hepatitis C infection. By way of example, a siRNA may beadministered in combination with interferon alpha. Additional HCVtreatments that may be used in combination with compositions of thepresent invention may include ribavirin and liver transplantation. It isalso contemplated that embodiments of the siRNAs, shRNAs, etc., of thepresent invention may be used in combination with other siRNAs or shRNAsdeveloped against CyPA or HCV targets and/or in combination withprotease inhibitors or polymerase inhibitors developed against HCV.

In addition, embodiments of siRNA and/or shRNA compositions of thepresent invention may be used in combination with Cyclosporin A (CsA) tomanage, treat, and/or prevent HCV infection. Embodiments of RNAicompositions of the present invention directed against CyPA may beparticularly advantageous because such compositions circumvent CsAresistance of some HCV strains to effectively inhibit such CsA-resistantHCV strains when administered in combination with CsA. In other words,treatment of cells infected or at risk of infection by a CsA-resistantstrain of HCV with embodiments of siRNA and/or shRNA compositions of thepresent invention may have the effect of “resensitizing” previouslyCsA-resistant HCV to CsA treatment. Thus, according to some embodiments,these compositions may provide a novel avenue for therapy against HCVwhen used in combination with an existing compound (CsA).

Methods

According to another embodiment of the present invention, methods areprovided for administering siRNA, shRNA, viral or non-viral vector, orDNA polynucleotide compositions of the present invention directedagainst CyPA (as described herein) to effectively inhibit HCV infectionin a cell or tissue. According to some embodiments, such methods may beused to prevent HCV infection in a cell or tissue at risk of HCVinfection, such as a liver cell or tissue. According to otherembodiments, such methods may be used to treat or manage HCV infectionin a cell or tissue infected with HCV. Such methods may compriseadministering such compositions in the form of a pharmaceuticalcomposition in combination with acceptable carriers, etc., oralternatively, such compositions may be administered in combination witha delivery reagent as described herein. In addition, such methods maycomprise administering such compositions directly to cells or tissuesinfected or at risk of infection by HCV without a delivery reagent asdescribed herein.

Embodiments of methods of the present invention may be further providedfor administering siRNA, shRNA, viral or non-viral vector, or DNApolynucleotide embodiments of compositions of the present inventiondirected against CyPA (as described herein) to effectively inhibit HCVinfection in an individual, subject, or patient. According to someembodiments, such methods may be used to prevent HCV infection in anindividual, subject, or patient at risk of HCV infection, such as anindividual, subject, or patient exposed to HCV. According to otherembodiments, such methods may be used to treat or manage HCV infectionin an individual, subject, or patient infected with HCV. Again, suchmethods may comprise administering such compositions in the form of apharmaceutical composition in combination with acceptable carriers,etc., or alternatively, such compositions may be administered incombination with a delivery reagent as described herein. In addition,such methods may comprise administering such compositions directly tocells or tissues infected or at risk of infection by HCV without adelivery reagent as described herein.

General Methods

Molecular biological techniques, biochemical techniques, andmicroorganism techniques as used herein are well known in the art andmay include, for example, Innis, M. A. et al., “PCR Strategies,”Academic Press (1995); Ausubel, F. M., “Short Protocols in MolecularBiology: A Compendium of Methods from Current Protocols in MolecularBiology,” (Wiley & Sons, 5^(th) Ed., 2002); Sninsky, J. J. et al., “PCRApplications: Protocols for Functional Genomics,” (Academic Press,1999); Sambrook J. et al., “Molecular Cloning: A Laboratory Manual,”(3^(rd) Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 2001); Freshney, R. I., “Culture of Animal Cells: A Manual ofBasic Techniques,” (4^(th) Ed., 2000); Spector, D. L., Cells: “ALaboratory Manual, Culture and Biochemical Analysis of Cells,” (ColdSpring Harbor Press, 1998); and the like. All relevant portions of eachof these references are hereby incorporated by reference. Geneintroduction may be confirmed by any standard method known in the art,such as those described herein, including, e.g., Northern blottinganalysis and Western blotting analysis, or other well-known, commontechniques. Any technique may be used herein for introduction of anucleic acid molecule into cells, including, for example,transformation, transduction, transfection, and the like. Such nucleicacid molecule introduction techniques are well known in the art andcommonly used.

EXAMPLES

The following non-limiting examples are provided to further illustratethe present invention. It should be appreciated by those of skill in theart that the techniques disclosed in the examples that follow representapproaches the inventors have found function well in the practice of theinvention, and thus can be considered to constitute examples of modesfor its practice. However, all examples in the present disclosure, whileillustrating embodiments of the present invention, are provided asnon-limiting examples and are, therefore, not to be taken as limitingthe various aspects of the present invention so illustrated. Those ofskill in the art should, in light of the present disclosure, appreciatethat many changes can be made in the specific embodiments that aredisclosed and still obtain a like or similar result without departingfrom the spirit and scope of the invention.

Example 1 Materials and Methods

Cells, Compounds, and Antibodies.

GS5 and RS2 cells have been described previously (36). Huh-7.5 cells andthe H77 replicon construct are provided by Charles Rice and Apath LLC.CsA is purchased from Alexis Corporation (San Diego, Calif.). Thefollowing antibodies are used: anti-CyPA (Biomol, Plymouth Meeting,Pa.), anti-CyPB (Affinity BioReagents, Golden, Colo.), anti-Ku80 andantiactin (Sigma-Aldrich), anti-NS5A and anti-NS5B (Virogen, Boston,Mass.), anti-NS3 (G. George Luo, University of Kentucky), and anticore(Affinity BioReagents, Golden, Colo.).

RNA Interference.

A human immunodeficiency virus (HIV)-based lentiviral vector is used toexpress all of the short hairpin RNAs (shRNAs). The sh-Luc and sh-B710RNAs have been described previously (Robida et al., “Characterization ofhepatitis C virus subgenomic replicon resistance to cyclosporine invitro,” J. Virol. 81:5829-5840.). Target or sense sequences for theother shRNAs are as follows:

A-159, 5′-AAG GGT TCC TGC TTT CAC AGA-3′ (SEQ ID NO: 6), whichcorrespond to nucleotides 159-179 of human CyPA cDNA (accession no.Y00052; RNA sequence for A-159 is provided in SEQ ID NO: 2);

A-285, 5′-AAG CAT ACG GGT CCT GGC ATC-3′, (SEQ ID NO: 7), whichcorrespond to nucleotides 285-305 of human CyPA cDNA;

A-285′, 5′-AAG CAT ACa GGT CCT GGC ATC-3′ (SEQ ID NO: 8), whichcorrespond to nucleotides 285-305 of human CyPA cDNA, with one mismatchrepresented in lower case;

A-459, 5′-AAT GGC AAG ACC AGC AAG AAG-3′ (SEQ ID NO: 9), whichcorrespond to nucleotides 459-479 of human CyPA cDNA;

C-454: 5′-AAG ACT GAA GGT GTG CTG GTA-3′ (SEQ ID NO: 10), whichcorrespond to nucleotides 454-474 of human CyPC cDNA (accession no.S71018); and

NTC, 5′-AAG GAG GTG ACA TCA CCA CTG-3′ (SEQ ID NO: 11), wherein NTC (notarget control) does not have a target in the human genome.

The above sequences may be converted to RNA sequences by changing eachthymine (T) to uracil (U). Lentiviral vector production and transductionare performed as described previously (44). Stable cells expressingshRNAs are obtained by selection with 1 μg/ml of puromycin (MPBiomedicals, Solon, Ohio) for 3 weeks.

In Vitro Transcription, Electroporation, Colony Formation Assays, andQuantitative RT-PCR.

The primers for detecting CyPA, CyPB, and CyPC have the followingsequences: A-Forward, 5′-CGG GTC CTG GCA TCT TGT-3′ (SEQ ID NO: 12) andA-Reverse, 5′-GCA GAT GAA AAA CTG GGA ACCA-3′ (SEQ ID NO: 13);B-Forward, 5′-GGC CAA CGC AGG CAA A-3′ (SEQ ID NO: 14) and B-Reverse,5′-TCT AGC CAG GCT GTC TTG ACT GT-3′ (SEQ ID NO: 15); C-Forward, 5′-GCTGAA GCA CTA TGG CAT TGG-3′ (SEQ ID NO: 16) and C-Reverse, 5′-GAA CTG AGAGCC ATT GGT GTC A-3′ (SEQ ID NO: 17).

Co-IP/RT-PCR.

Replicon cells (5×10⁶) are seeded into a T-75 flask (treated with 4μg/ml CsA where indicated) 1 day before the immunoprecipitation (IP)experiment. Twenty-four hours later, cells are lysed in 1 ml of IPbuffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM phenylmethylsulfonylfluoride [PMSF], and 0.5% NP-40). Two hundred units of RNaseOUT(Invitrogen, Carlsbad, Calif.) are added to the supernatant aftercentrifugation at 12,000×g for 15 min. The supernatant is then added to50 μl 75% protein G slurry containing either anti-CyPA or rabbitimmunoglobulin G (IgG). The binding is allowed to proceed at 4° C.overnight, after which the protein G beads are washed with IP bufferfour times. RNA is extracted from the beads with an RNeasy kit (Qiagen,Valencia, Calif.). RT-PCR is then used to detect HCV internal ribosomeentry site (IRES) with the following primers: IRES Forward, 5′-GTC TGCGGA ACC GGT GAG-3′; (SEQ ID NO: 18); IRES Reverse, 5′-CGG GTT GAT CCAAGA AAG GAC-3′ (SEQ ID NO: 19).

Recombinant Protein Production and GST Binding Assay.

Recombinant protein expression and purification via glutathioneSepharose 4B beads are carried out according to the manufacturer'sprotocol (GE Healthcare, Piscataway, N.J.). For the glutathioneS-transferase (GST) pull-down assay, 20 μg of GST or GST-CyPA is broughtto a final volume of 200 μl with binding buffer (20 mM Tris-HCl, pH 7.5,100 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 5 mM dithiothreitol [DTT], 0.5%NP-40, 0.5 mM PMSF, 5% glycerol). Replicon cells (4×10⁵) are lysed in300 μl of IP buffer supplemented with 1 mM DTT and 1 mM EDTA, and 40 μlof this lysate is added to each sample and allowed to rotate at 4° C.for 30 min. Glutathione Sepharose 4B beads (25 μl of a 50% slurry persample) are then added to each sample and allowed to rotate at 4° C. for30 min. Beads are washed and then sedimented at 500×g for 5 min.Proteins bound to the beads are analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Westernblotting. For the reactions that included CsA, the compound is added tothe recombinant proteins prior to the addition of the cell lysates.

Co-IP of NS5B and CyPA.

293-T cells are transfected with cDNAs expressing Con1 NS5B andFlag-CyPA for 48 h. Cells are lysed in IP buffer (50 mM Tris-HCl, pH8.0, 150 mM NaCl, 0.5% NP-40,1 mM PMSF, 1 mM EDTA, 1 mM DTT, 1× proteaseinhibitor cocktail) by rotating at 4° C. for 30 min. The lysate is thenclarified by centrifugation, and the supernatant is subjected to IP withEZview anti-Flag M2 affinity beads (Sigma-Aldrich, Mo.) according to themanufacturer's instructions. Proteins bounds to the beads following theIP protocol are eluted by boiling in SDS loading buffer and analyzed bySDS-PAGE followed by Western blotting using the indicated antibodies.

JFH-1/HCVcc Production and Infection.

Full-length JFH-1 cDNA is provided by Takaji Wakita. Production ofinfectious HCVcc and infection of Huh-7.5 cells are performed asdescribed previously (See, e.g., Zhong, J. et al., “Robust hepatitis Cvirus infection in vitro,” Proc. Natl. Acad. Sci. USA, 102:9294-9299(2005)). Western blotting and immunostaining of infected cells arecarried out according to standard methods.

Electroporation of Replicon RNA and cDNA Expression Plasmids.

One microgram of replicon RNA is mixed with 9 μg of a pcDNA3.1-basedplasmid containing no insert, CyPA cDNA, CyPB cDNA, or CyPC cDNA andused in a standard electroporation (36). RNA is extracted 7 h and 4 dayspost-electroporation and subjected to quantitative RT-PCR analysis todetect HCV RNA as described previously (See, e.g., Robida, J. M et al.,Characterization of hepatitis C virus subgenomic replicon resistance tocyclosporine in vitro, J. Virol., 81:5829-5840 (2007). All the CyP cDNAsare tagged with a Myc epitope at the C terminus, and the CyPA cDNAcontaining silent mutations in the sh-A159 recognition site isdesignated CyPA#.

In Vitro Replication Assay.

Replicon cells are washed with ice-cold wash buffer (30 mM HEPES [pH7.4], 33 mM NH4C1, 7 mM KCl, 150 mM sucrose, 4.5 mM magnesium acetate)containing freshly added lysolecithin (250 μg/ml) for 2 min. Aftercomplete aspiration of the wash buffer, 125 p. 1 of incompletereplication buffer (100 mM HEPES [pH 7.4], 50 mM NH4C1, 7 mM KCl, 1 mMspermidine) is added to each plate, and the cell lysates are collectedwith a cell scraper. The lysate is then centrifuged at 800×g for 5 minto remove cell debris, and the recovered supernatant is stored at −80°C. as replication lysates, 70 p. 1 of which is used for each in vitroreplication assay as described below. Before the replication assay isperformed, NP-40 (1%, vol/vol) is added to each replication lysate andthe mixture is rotated at 4° C. for 1 h, after which time 7 p. 1 rabbitanti-CyPA polyclonal antibody or normal rabbit IgG (negative control) isadded, and the mixture is rotated at 4° C. for an additional 4 h. Invitro replication is carried out for 4 h at 4° C. in the presence ofactinomycin D (10 μg/ml); RNaseOUT (800 U/ml); 25 μCi α-³²P-labeled CTP;10 μM CTP; and 1 mM each of ATP, GTP, and UTP. RNA is immediatelyextracted after replication with TRIzol LS (Invitrogen) as described bythe manufacturer. The products of the reactions are electrophoresed on1.2% agarose gels for 2 to 3 h, dried on a gel dryer, and exposed to aphosphorimaging screen for at least about 24 h.

Example 2 A Small Interfering RNA (siRNA) Directed at Inhibiting CyPAWild-Type But not CsA-Resistant HCV Replicons

A HCV subgenomic replicons resistant to CsA treatment is isolated invitro (see, Robida, J. M. et al., “Characterization of hepatitis C virussubgenomic replicon resistance to cyclosporine in vitro,” J. Virol.81:5829-5840 (2007)), the entire disclosure and contents of which ishereby incorporated by reference). Cells containing the RS2 replicon areresistant to CsA at up to 2 μg/ml, while the cells expressing the GS5cells are inhibited by CsA at even 0.25 μg/ml (FIG. 1A). To investigatethe mechanism of this resistance, the dependence of these replicons onthe three CyPs (CyPA, -B, and —C) that have been implicated in HCVreplication is examined The shRNAs expressed are directed at each ofthese CyPs along with a control shRNA directed at firefly luciferase ineither the GS5 or RS2 cells. All three CyP-directed shRNAs efficientlyknocked down the expressions of their respective targets (FIGS. 1B andC). The shRNA directed at CyPA (sh-A159) inhibits NS5A expression in GS5cells but not in RS2 cells, despite similar knock-down levels of CyPA inthe two cell lines. The shRNAs directed at CyPB (si-B710) or CyPC(sh-C454) has no effect on the NS5A level in either GS5 or RS2 cells.Fluorescence-activated cell sorting (FACS) results with greenfluorescent protein (GFP) expression as a readout confirms theseresults. The same panel of shRNAs is also used in a colony formationassay designed to test their effects on HCV replication. The repliconcells are transduced with shRNA-expressing vectors that carry thepuromycin N-acetyltransferase (pac) gene and then the cells aresubjected to a double selection with both puromycin and G418, whichselects for cells that maintain replication. When GS5 cells aretransduced with sh-A159, a significantly lower number ofdouble-resistant colonies are observed, reflecting the inhibitory effectof sh-A159 on GS5 replication. This lower colony formation efficiency isnot observed in RS2 cells (FIG. 1D). On the other hand, shRNAs directedat CyPB and CyPC has no significant effect on the number of colonies.Similar results are obtained with a CsA-resistant replicon single-cellclone, RS1-2.

To further validate a role of CyPA protein in HCV replication, in vitroreplication assays are performed with cell extracts of replicon cellsand then tested the ability of an antibody against CyPA to interferewith replication. Anti-CyPA effectively blocks replication when theassay is performed with GS5 lysate but fails to inhibit replication ifRS2 lysate is used (FIG. 1E). These data corroborate the RNAinterference results and suggest that CyPA protein is important for astep in the HCV replication process that may be measured by the in vitroreplication assay.

Example 3 HCV Replicon Expression is Correlated with CyPA ExpressionLevel

Several other siRNAs directed at CyPA have been evaluated in vitro foranti-HCV effects. However, these studies have yielded conflictingresults. Although two shRNAs directed at CyPA are reported to have bothknocked down CyPA and inhibited replication of a GT 1b replicon. See,Nakagawa, M. et al., “Suppression of hepatitis C virus replication bycyclosporine a is mediated by blockade of cyclophilins,”Gastroenterology 129:1031-1041 (2005). A CyPA siRNA that efficientlysuppressed CyPA expression but fails to inhibit HCV replication is alsoreported. See, Watashi, K. et al., “Cyclophilin B is a functionalregulator of hepatitis C virus RNA polymerase,” Mol. Cell. 19:111-122(2005).

In an attempt to reconcile these results and to develop a more robustsiRNA composition against CyPA and HCV, shRNAs is constructed directedat the same CyPA mRNA sites as reported by these groups (FIG. 2A) andcompares their effects on HCV replication to an embodiment of sh-A159herein. The sh-285's target is the same 21-nucleotide sequence asWatashi's siRNA (“si-CyPA”) and sh-459's is a 21-nucleotide sequencethat is encompassed by Nakagawa's shRNA #441, which has a targetsequence of 29 nucleotides. An sh-A285′ is also constructed, which isidentical to sh-285 except for a mismatch of one nucleotide between thesiRNA and the target sequence. Consequently, sh-285′ is expected to lackthe ability to knock down CyPA. Finally, a negative-control shRNA(sh-NTC) is constructed that does not recognize any human mRNA inGenBank. When this panel of shRNAs is introduced into GS5 cells, threeof them, sh-A159, sh-A285, and sh-A459, knock down CyPA expression as isexpected (FIG. 2B). All three cell lines with CyPA knock-downs also havereduced NS5A levels. The shRNAs that do not inhibit CyPA expression alsodo not affect the replicon. Importantly, however, the original shRNA(sh-A159) embodiment is the most effective in both silencing CyPAexpression and inhibiting NS5A synthesis (FIG. 2B).

Because the different effects of sh-A285 and Watashi's si-CyPA couldpotentially be explained by differences between shRNA and synthesizedsiRNA duplex, a synthesized form of sh-A159 and sh-A285 is tested intransient transfection experiments. Both siRNAs (si-A159 and si-A285)inhibited CyPA and NS5A expression (FIG. 2C). In this form, si-A159 isno more potent than si-A285 in suppressing CyPA expression; accordingly,the abilities of these two siRNAs to inhibit HCV replicon are alsocomparable. The overall reduction of both CyPA and NS5A levels is lessdramatic with these preformed siRNA duplexes, likely due to the lowerefficiency of transfecting siRNA into cells in comparison to transducingthe shRNAs using lentiviral vectors. To eliminate definitively thepossibility that the target of all these siRNAs is a chimeric mRNAcontaining the entire CyPA mRNA as part of its sequence (See, e.g.,Sayah, D. M. et al., “Cyclophilin A retrotransposition into TRIMSexplains owl monkey resistance to HIV-1,” Nature 430:569-573 (2004)),cDNA rescue experiments are performed. A cDNA of CyPA that containssilent mutations in the recognition site of sh-A159 is constructed andcloned into a mammalian expression vector. A Myc tag is placed at theC-terminus of the protein to allow specific detection. When this cDNA(Myc-A#) is introduced into replicon cells together with sh-A159, bothNS5A expression (FIG. 2D) and HCV RNA replication (FIG. 2E) arepartially rescued, indicating that sh-A159 indeed exerts its inhibitoryeffect by repressing CyPA expression.

Example 4 CyPA Mediates CsA Resistance of RS2 Cells

In contrast to the GS5 replicon, RS2 replicates efficiently in thepresence of substantial CyPA knock-down (FIG. 1D). This result could beexplained by either CyPA-independent replication or a replicationstrategy that requires much less CyPA protein because shRNAs normallycannot eliminate the gene product completely. To distinguish betweenthese two possibilities, the RS2/sh-A159 replicon cells are treated, andrecovered after double selection of puromycin and G418 following shRNAtransduction, with CsA. If RS2 replicates in a CyP-independent manner inthese cells, one would expect no effect of the CsA on the RS2/sh-A159cells. On the other hand, if the RS2 replicon is resistant to CsA andsh-A159 because it needs less CyPA to replicate (thus requiring a higherconcentration of CsA to inhibit its replication), then one would expecta reduced resistance to CsA as the pool of CyPA is smaller in this case.Indeed, the latter appears to be the case, as the RS2/sh-A159 repliconis six to eight times more sensitive to CsA than is the RS2/sh-Lucreplicon (FIG. 3A), essentially reverting to the sensitivity level ofthe wild-type replicon GS5. Western blotting confirmed the substantialsuppression of CyPA in the RS/sh-A159 cells (FIG. 3B). To control forthe possibility that the difference in sensitivity to CsA is caused bysome unidentified differences in the antibiotic-selected cells, a doubletreatment (sh-A159 and CsA) is applied to RS2 cells without selection.When the RS2 cells are treated with 0.5 μg/ml CsA or are transduced withsh-A159, no significant suppression is seen in either treatment, butwhen RS2 cells are first transduced with sh-A159 and are then treatedwith the same concentration of CsA, a dramatic inhibition is achieved(FIG. 3C). Taken together, these data indicate that CyPA is theprincipal mediator of the CsA resistance observed in RS2 cells, thereplication of which requires a much reduced level of CyPA.

Example 5 CyPA is Essential for the Replication of Multiple HCV Isolates

GS5 and RS2 cells contain GT 1b replicons with the GFP gene insertedinto the NS5A region. (See, e.g., Moradpour, D. et al., “Insertion ofgreen fluorescent protein into nonstructural protein 5A allows directvisualization of functional hepatitis C virus replication complexes,” J.Virol. 78:7400-7409 (2004); Nelson, H. B. et al., “Effect of cell growthon hepatitis C virus (HCV) replication and a mechanism of cellconfluence-based inhibition of HCV RNA and protein expression,” J.Virol. 80:1181-1190 (2006)). Next the effect of CyPA knock-down onreplicons without GFP is examined. Both GT 1a and 1b replicons aretested. To this end, stable Huh-7.5 cell lines are established thatexpress the various shRNAs and then challenges them, by electroporation,with in vitro-transcribed GT 1a (H77) and 1b (Con1) subgenomic repliconRNAs. The shRNAs effectively silence the expression of their respectivetargets in the stable cells as expected (FIGS. 4A and 4B). No defect inmorphology or growth rate is detected for any of the stable cell lines,confirming that these CyPs are dispensable for cell survival in vitro.See, e.g., Braaten, D. et al., “Cyclophilin A regulates HIV-1infectivity, as demonstrated by gene targeting in human T cells,” EMBOJ. 20:1300-1309 (2001). Expression of sh-A159 completely inhibits theability of either replicon RNA to form colonies, whereas sh-Luc has noeffect (FIG. 4C). An inhibitory effect (−50%) of sh-B710 on the GT 1areplicon is sometimes observed, but the colony formation efficiency isstill much higher than that in sh-A159 cells. No other shRNA shows anyconsistent inhibitory effect on any of the replicons.Transient-transduction experiments again confirm the inhibitory effectof sh-A159 on the expression of NS5A of both GT 1a and 1b replicons(FIG. 4D).

The complete cycle of HCV infection can now be studied in cell culturewith infectious viruses produced in vitro (see, e.g., Cai, Z. et al.,“Robust production of infectious hepatitis C virus (HCV) from stably HCVcDNA-transfected human hepatoma cells,” J. Virol. 79:13963-13973 (2005);Lindenbach, B. D. et al., “Complete replication of hepatitis C virus incell culture,” Science 309:623-626 (2005); Wakita, T. et al.,“Production of infectious hepatitis C virus in tissue culture from acloned viral genome,” Nat. Med. 11:791-796 (2005); Yi, M. et al.,“Production of infectious genotype 1a hepatitis C virus (Hutchinsonstrain) in cultured human hepatoma cells,” PNAS 103:2310-2315 (2006);Zhong, J. et al., “Robust hepatitis C virus infection in vitro,” PNAS102:9294-9299 (2005)), the entire disclosures and contents of which arehereby incorporated by reference), so whether CyPA is required for HCVinfection in vitro is determined next. Stable Huh-7.5 cells harboringcontrol or sh-A159 are infected with HCVcc particles which are producedwith the JFH-1 genome. Infection of Huh-7.5 cells is efficient; viralRNA and antigens become readily detectable in the target cells within afew days after infection. Expression of sh-Luc or sh-B710 has no effecton HCV infection, whereas the sh-A159 cells are highly refractory toinfection (FIG. 5A). The protection provided by sh-A159 is observed byseveral methods: RT-PCR, fluorescence staining for core protein, orWestern blot detection of NS3 (FIGS. 5B and C). The sh-A159 cellsremains fully susceptible to infection by vesicular stomatitis virus, anegative-strand RNA virus that is sensitive to nonspecific antiviralresponses (FIG. 5D).

Last, to determine whether sh-A159 could repress an existing infection,Huh-7.5 cells are infected with JFH-1 virus for 10 days and are thenintroduced sh-A159 by transduction. The expression of NS3 is measured 7days after transduction. As shown in FIG. 5E, delivery of sh-A159 intoinfected cells suppresses viral replication, parallel to the resultswhich are obtained with transient transduction of replicon cells.

Example 6 The Association of CyPA with HCV Polymerase and RNA inReplicon Cells is Correlated with CsA Resistance

In vitro replication results suggests that CyPA is directly involved inthe replication process (FIG. 1E). To determine whether CyPA isassociated with HCV genome in replicon cells, CyPA are precipitated fromreplicon lysates and are extracted the coprecipitated RNA, which is thensubjected to RT-PCR analysis with primers complementary to the 5′nontranslated region of HCV. HCV RNA is found to be precipitated by ananti-CyPA antibody but not by an IgG control antibody (FIG. 6A). Thisassociation is inhibited by CsA treatment when the experiment isperformed with the GS5 replicon (FIG. 6B, left). For the RS2 replicon,association with CyPA is resistant to CsA treatment (FIG. 6B, right).The interaction between CyPA and the HCV polymerase NS5B is examinednext. GST-CyPA specifically binds NS5B while GST protein alone is notable to bind (FIG. 6C). The interaction between GS5 NS5B and GST-CyPA isreduced by CsA treatment and becomes abolished at 2.4 μg/ml CsA (FIG.6D, left). NS5B from the RS2 cells, however, retains binding to GST-CyPAeven at this concentration of the drug (FIG. 6D, right). These resultssuggest that the association of CyPA with the HCV replication machineryis targeted by CsA and the CsA-resistant interaction between NS5B andCyPA contributes to the CsA-resistant replication of the RS2 replicon.To examine if NS5B could interact with CyPA in vivo in the absence ofany other HCV proteins and viral RNA, co-IP experiments are performedwith NS5B and Flag-tagged CyPA transiently expressed in 293-T cells.NS5B coprecipitates with CyPA in this setting (FIG. 6E), indicating thatthe CyPA-NS5B interaction in vivo is not mediated by any other viralprotein or RNA. Only a fraction of the total NS5B is precipitated by theanti-Flag beads as expected because the expressed NS5B proteins areexpected to interact with both Flag-tagged and untagged, endogenousCyPA.

Example 7 Different Expression Levels of CyP Isoforms in Replicon Cells

Consistent with the critical role of CyPA in mediating CsA's action inregulating a variety of biological activities such as immunosuppression(see, e.g., Handschumacher, R. E. et al., “Cyclophilin: a specificcytosolic binding protein for cyclosporine A,” Science 226:544-547(1984)), HIV infection (see, e.g., Braaten, D. et al., “Cyclophilin Aregulates HIV-1 infectivity, as demonstrated by gene targeting in humanT cells,” EMBO J. 20:1300-1309 (2001)), and HCV replication, it has beenshown that the expression level of CyPA is 10 to 100 times higher thanthat of other CyPs in various tissues (see, e.g., Bergsma, D. J. et al.,“The cyclophilin multigene family of peptidyl-prolyl isomerases:Characterization of three separate human isoforms,” J. Biol. Chem.266:23204-23214 (1991)). The endogenous expression level of the threeCyP isoforms in the replicon cells is examined Using quantitative RT-PCRand glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as an internalcontrol, a significant difference in the expression levels of CyPA,CyPB, and CyPC in the replicon cells is discovered. The CyPA mRNA isexpressed approximately 10 and 150 times higher than CyPB and CyPCmRNAs, respectively (FIG. 7). Moreover, the expression levels of theCyPs are found to be very similar in GS5 and RS2 cells, ruling out thepossibility that RS2 cells are more resistant to CsA because of higherendogenous level of CyPs.

For further discussion of various examples in the present applications,see, e.g., Yang, F. et al., “Cyclophilin A is an essential cofactor forhepatitis C virus infection and the principal mediator of cyclosporineresistance in vitro,” J. Virology 82(11):5269-78 (2008), the entiredisclosure and contents of which is hereby incorporated by reference.

All documents, patents, journal articles and other materials cited inthe present application are hereby incorporated by reference in theirentirety. Although the present invention has been fully described inconjunction with several embodiments thereof with reference to theaccompanying drawings, it is to be understood that various changes andmodifications may be apparent to those skilled in the art. Such changesand modifications are to be understood as included within the scope ofthe present invention as defined by the appended claims, unless theydepart therefrom.

1. A DNA polynucleotide comprising a DNA sequence region encoding asense RNA sequence that is at least about 70% homologous to at least 19contiguous nucleotides between nucleotides 155 and 183 of humancyclophilin A sequence (SEQ ID NO: 1), wherein the sense RNA strand isfrom 19 to 29 nucleotides in length; further comprising a DNA sequenceregion encoding an antisense RNA sequence that is complementary to thesense RNA sequence; and wherein the DNA polynucleotide isdouble-stranded and the DNA sequence region encoding a sense RNAsequence and the DNA sequence region encoding an antisense RNA sequenceare base-paired with each other on opposing strands.
 2. (canceled) 3.(canceled)
 4. (canceled)
 5. A plasmid vector, comprising the DNApolynucleotide of claim
 1. 6. The plasmid vector of claim 5, furthercomprising a promoter, wherein the DNA sequence region encoding a senseRNA sequence is under the control of the promoter.
 7. The plasmid vectorof claim 6, wherein the promoter comprises an inducible promoter.
 8. Theplasmid vector of claim 6, wherein the promoter comprises atissue-specific promoter.
 9. The plasmid vector of claim 6, wherein thetissue-specific promoter comprises an albumin promoter.
 10. The plasmidvector of claim 6, wherein the promoter comprises an RNA polymerase IIIpromoter.
 11. The plasmid vector of claim 10, wherein the RNA polymeraseIII promoter is selected from the group consisting of H1, U6, 5S, 75K,and tRNA promoters.
 12. A delivery composition, comprising the DNApolynucleotide of claim 1 in combination with a delivery reagent. 13.The delivery composition of claim 12, wherein the delivery reagent isselected from the group consisting of Minis Transit lipophilic reagent,lipofectin, lipofectamine, cellfectin, cationic polymers, cationiclipids, and liposomes.
 14. The DNA polynucleotide of claim 1, furthercomprising a DNA sequence region encoding a hairpin sequence insertedbetween the DNA sequence region encoding a sense RNA sequence and theDNA sequence region encoding an antisense RNA sequence, such that thesense RNA sequence encoded by the DNA polynucleotide and the antisenseRNA sequence encoded by the DNA polynucleotide are covalently attachedby the hairpin sequence to form a shRNA.
 15. The DNA polynucleotide ofclaim 14, wherein the shRNA encoded by the DNA polynucleotide comprisesSEQ ID NO:
 3. 16. A plasmid vector, comprising the DNA polynucleotide ofclaim
 14. 17. The plasmid vector of claim 16, further comprising apromoter, wherein the DNA sequence regions encoding the shRNA are underthe control of the promoter.
 18. The plasmid vector of claim 17, whereinthe promoter comprises an inducible promoter.
 19. The plasmid vector ofclaim 17, wherein the promoter comprises a tissue-specific promoter. 20.The plasmid vector of claim 17, wherein the tissue-specific promotercomprises an albumin promoter.
 21. The plasmid vector of claim 17,wherein the promoter comprises an RNA polymerase III promoter.
 22. Theplasmid vector of claim 21, wherein the RNA polymerase III promoter isselected from the group consisting of H1, U6, 5S, 75K, and tRNApromoters.
 23. A delivery composition, comprising the DNA polynucleotideof claim 14 in combination with a delivery reagent.
 24. The deliverycomposition of claim 23, wherein the delivery reagent is selected fromthe group consisting of Minis Transit lipophilic reagent, lipofectin,lipofectamine, cellfectin, cationic polymers, cationic lipids, andliposomes.
 25. (canceled)
 26. (canceled)
 27. (canceled)
 28. (canceled)29. (canceled)
 30. (canceled)
 31. (canceled)